Compositions and methods for detecting zika virus nucleic acid

ABSTRACT

Disclosed are nucleic acid oligomers, including amplification oligomers, capture probes, and detection probes, for detection of Zika virus nucleic acid. Also disclosed are methods of specific nucleic acid amplification and detection using the disclosed oligomers, as well as corresponding reaction mixtures and kits.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/659,375, filed Oct. 21, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/618,834, filed Jun. 9, 2017, now issued as U.S.Pat. No. 10,508,312, which claims benefit of priority under 35 U.S.C §119(e) to U.S. Provisional Application No. 62/348,563, filed Jun. 10,2016, each of which is incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII Copy, created on Sep. 25, 2020, isnamed “DIA-0025-05-DV 1_SeqList_ST25.txt” and is 57,994 bytes in size.

BACKGROUND

Zika virus is a mosquito-borne Flavivirus that has been associated withhuman disease ranging from subclinical to mild illnesses. Clinicalcharacteristics of Zika virus infection include fever, headache,malaise, stomach ache, dizziness, anorexia, and maculopapular rash Zikavirus infection has also been associated with serious and sometimesfatal cases of Guillain-Barré syndrome. Additionally, there is mountingevidence indicating that Zika virus infection can cause microcephaly andother birth defects in infants born to infected mothers. Although theprimary route of infection is through the bite of a mosquito, sexualtransmission and possible transfusion transmission of Zika virus havebeen reported.

SUMMARY

Disclosed herein is a combination of at least two amplificationoligomers for amplifying a Zika virus nucleic acid in a sample, theoligomer combination comprising: at least two amplification oligomersfor amplifying a target sequence of a Zika virus target nucleic acid,wherein the target sequence consists essentially of SEQ ID NO:189,accounting for complements thereof, RNA equivalents thereof, or both,wherein the first amplification oligomer and the second amplificationoligomer are configured to hybridize to opposite ends of the targetsequence, to generate amplification products; wherein a firstamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 26 contiguous nucleobases in length, and wherein a secondamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 26 contiguous nucleobases in length and, optionally, joinedat its 5′ end to a promoter sequence. In some aspects of the combinationof at least two amplification oligomers, the first amplificationoligomer is contained within SEQ ID NO:24 and contains SEQ ID NO:25. Insome aspects, the first amplification oligomer comprises a sequence thatis selected from the group consisting of: SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17,SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:49, SEQ ID NO:50,SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53. In some aspects, thetarget hybridizing region of the second amplification oligomer iscontained within SEQ ID NO:150. In some aspects, the target hybridizingregion of the second amplification oligomer is 19 contiguous nucleobasesin length or is 20 contiguous nucleobases in length. In some aspects,the target hybridizing sequence of the second amplification oligomer isselected from the group consisting of: SEQ ID NO:112, SEQ ID NO:113, SEQID NO:114, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:125, SEQ ID NO:126,SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ IDNO:131, and SEQ ID NO:134. In some aspects, the second amplificationoligomer is joined at its 5′ end to a promoter sequence that is a T7promoter sequence. In some aspects, the T7 promoter sequence consists ofSEQ ID NO:179. In some aspects, the second amplification oligomercomprises a sequences selected from the group consisting of: SEQ IDNOs:74 to 89, SEQ ID NO:107, and SEQ ID NO:108.

In some aspects of the combination of at least two amplificationoligomers, the combination of first amplification oligomer and secondamplification oligomer comprise nucleic acid sequences selected from oneof the following groups (i) SEQ ID NO:16 and SEQ ID NO:78; (ii) SEQ IDNO:16 and SEQ ID NO:80; (iii) SEQ ID NO:16 and SEQ ID NO:75; (iv) SEQID NO:16 and SEQ ID NO:89; (v) SEQ ID NO:16 and SEQ ID NO:74; (vi) SEQID NO:11 and SEQ ID NO:78; (vii) SEQ ID NO:11 and SEQ ID NO:80; (viii)SEQ ID NO:11 and SEQ ID NO:75; (ix) SEQ ID NO:11 and SEQ ID NO:89; (x)SEQ ID NO:11 and SEQ ID NO:74; (xi) SEQ ID NO:12 and SEQ ID NO:79; (xii)SEQ ID NO:12 and SEQ ID NO:87; (xiii) SEQ ID NO:12 and SEQ ID NO:89;(xiv) SEQ ID NO:12 and SEQ ID NO:74; (xv) SEQ ID NO:17 and SEQ ID NO:79;(xvi) SEQ ID NO:17 and SEQ ID NO:87; (xvii) SEQ ID NO:17 and SEQ IDNO:89; (xviii) SEQ ID NO:17 and SEQ ID NO:74; (xix) SEQ ID NO:53 and SEQID NO:78; (xx) SEQ I DNO:53 and SEQ ID NO:80; (xxi) SEQ ID NO:53 and SEQID NO:75; (xxii) SEQ ID NO:53 and SEQ ID NO:89; (xxiii) SEQ ID NO:53 andSEQ ID NO:74; (xxiv) SEQ ID NO:18 and SEQ ID NO:78; (xxv) SEQ I DNO:18and SEQ ID NO:80; (xxvi) SEQ ID NO:18 and SEQ ID NO:75; (xxvii) SEQ IDNO:18 and SEQ ID NO:89; or (xxviii) SEQ ID NO:18 and SEQ ID NO:74.

In some aspects of the combination of at least two amplificationoligomers, the combination comprises a third amplification oligomer foramplifying a target sequence of a Zika virus nucleic acid, wherein thethird amplification oligomer comprises a target hybridizing sequenceselected from the group consisting of: SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, and SEQ ID NO:53, and wherein the targethybridizing sequence of the first amplification oligomer and the targethybridizing sequence of the third amplification oligomer are differentsequences. In some aspects, the combination of first amplificationoligomer, second amplification oligomer and third amplification oligomeris one of: (i) a first amplification oligomer comprising a nucleic acidsequence consisting of SEQ ID NO:16; a second amplification oligomercomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NOs:74, 75, 78, 80, & 89; and a third amplification oligomercomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NOs:11, 12, 17, 18, & 53; (ii) a first amplification oligomercomprising a nucleic acid sequence consisting of SEQ ID NO:11; a secondamplification oligomer comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs:74, 75, 78, 80, & 89; and a thirdamplification oligomer comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs:12, 17, 18, & 53; (iii) a firstamplification oligomer comprising a nucleic acid sequence consisting ofSEQ ID NO:12; a second amplification oligomer comprising a nucleic acidsequence selected from the group consisting of SEQ ID NOs:74, 75, 78,80, & 89; and a third amplification oligomer comprising a nucleic acidsequence selected from the group consisting of SEQ ID NOs:17, 18, & 53;(iv) a first amplification oligomer comprising a nucleic acid sequenceconsisting of SEQ ID NO:17; a second amplification oligomer comprising anucleic acid sequence selected from the group consisting of SEQ IDNOs:74, 75, 78, 80, & 89; and a third amplification oligomer comprisinga nucleic acid sequence selected from the group consisting of SEQ IDNOs:18, & 53; or (v) a first amplification oligomer comprising a nucleicacid sequence consisting of SEQ ID NO:18; a second amplificationoligomer comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs:74, 75, 78, 80, & 89; and a third amplificationoligomer comprising a nucleic acid sequence consisting of SEQ ID NO:53.In some aspects of the combination of at least two amplificationoligomers, the combination comprises a fourth amplification oligomer foramplifying a target sequence of a Zika nucleic acid, wherein the fourthamplification oligomer comprises a target hybridizing sequence selectedfrom the group consisting of: SEQ ID NO113, SEQ ID NO:114, SEQ IDNO:115, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:128, & SEQ ID NO:134,and wherein the target hybridizing sequence of the fourth amplificationoligomer is optionally joined at its 5′ end to a promoter sequence, andwherein the target hybridizing sequence of the second amplificationoligomer and the target hybridizing sequence of the fourth amplificationoligomer are different sequences. In some aspects, the targethybridizing sequence of the fourth amplification oligomer is joined to apromoter sequence is a T7 promoter sequence. In some aspects, thepromoter sequence consists of SEQ ID NO:179. In some aspects, the fourthamplification oligomer comprises a sequence that is selected from thegroup consisting of: SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:78, SEQ IDNO:79, SEQ ID NO:80, SEQ ID NO:87, and SEQ ID NO:89.

Further disclosed herein is a combination of at least two amplificationoligomers for amplifying a Zika virus nucleic acid in a sample, theoligomer combination comprising: at least two amplification oligomersfor amplifying a target sequence of a Zika virus target nucleic acid,wherein the target sequence consists essentially of SEQ ID NO:187,accounting for complements thereof, RNA equivalents thereof, or both,wherein the first amplification oligomer and the second amplificationoligomer are configured to hybridize to opposite ends of the targetsequence, including complements thereof and/or RNA equivalents thereof,to generate amplification products; wherein a first amplificationoligomer comprises a target hybridizing sequence that is from 19 to 23contiguous nucleobases in length, and wherein a second amplificationoligomer comprises a target hybridizing sequence that is from 19 to 25contiguous nucleobases in length and, optionally, joined at its 5′ endto a promoter sequence.

In some aspects of the combination of at least two amplificationoligomers, the target hybridizing sequence of the first amplificationoligomer is contained within SEQ ID NO:4. In some aspects, the targethybridizing sequence of the first amplification oligomer contains SEQ IDNO:2. In some aspects, the first amplification oligomer comprises asequence selected from the group consisting of: SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and SEQID NO:35. In some aspects, the target hybridizing sequence of the secondamplification oligomer is contained within SEQ ID NO:152. In someaspects, the target hybridizing sequence of the second amplificationoligomer contains SEQ ID NO:148. In some aspects, the target hybridizingsequence of the second amplification oligomer contains SEQ ID NO:149. Insome aspects, the second amplification oligomer is joined at its 5′ endto a promoter sequence that is a T7 promoter sequence. In some aspects,the T7 promoter sequence consists of SEQ ID NO:179. In some aspects, thetarget hybridizing sequence of the second amplification oligomer isselected from the group consisting of: SEQ ID NO:111, SEQ ID NO:116, SEQID NO:117, SEQ ID NO:122, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137,and SEQ ID NO:138. In some aspects, the second amplification oligomercomprises a sequence selected from the group consisting of: SEQ IDNO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ IDNO:95, SEQ ID NO:96, and SEQ ID NO:97.

In some aspects of the combination of at least two amplificationoligomers, the combination of first amplification oligomer and secondamplification oligomer comprise nucleic acid sequences selected from oneof the following groups: (i) SEQ ID NO:30 and SEQ ID NO:92; (ii) SEQ IDNO:30 and SEQ ID NO:94; (iii) SEQ ID NO:30 and SEQ ID NO:95 (iv) SEQ IDNO:35 and SEQ ID NO:92; (v) SEQ ID NO:35 and SEQ ID NO:94; and vi) SEQID NO:35 and SEQ ID NO:95. In some aspects, the combination furthercomprises a third amplification oligomer for amplifying a targetsequence of a Zika virus nucleic acid, wherein the third amplificationoligomer comprises a target hybridizing sequence selected from the groupconsisting of: SEQ ID NO:111, SEQ ID NO:116, SEQ ID NO:117, SEQ IDNO:122, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, & SEQ ID NO:138,wherein, optionally, the target hybridizing sequence of the thirdamplification oligomer is joined at it 5′ end to a promoter sequence,and wherein the target hybridizing sequence of the second amplificationoligomer and the target hybridizing sequence of the third amplificationoligomer are different sequences. In some aspects, the targethybridizing sequence of the third amplification oligomer is joined atits 5′ end to a promoter sequence that is a T7 promoter sequence. Insome aspects, the promoter sequence consists of SEQ ID NO:179. In someaspects, the third amplification oligomer comprises a sequence selectedfrom the group consisting of: SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92,SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, and SEQ IDNO:97.

Further disclosed herein is a combination of at least two amplificationoligomers for amplifying a Zika virus nucleic acid in a sample, theoligomer combination comprising: at least two amplification oligomersfor amplifying a target sequence of a Zika virus target nucleic acid,wherein the target sequence consists essentially of SEQ ID NO:188,accounting for complements thereof, RNA equivalents thereof, or both,wherein the first amplification oligomer and the second amplificationoligomer are configured to hybridize to opposite ends of the targetsequence, including complements thereof and/or RNA equivalents thereof,to generate amplification products; wherein a first amplificationoligomer comprises a target hybridizing sequence that is from 19 to 23contiguous nucleobases in length, and wherein a second amplificationoligomer comprises a target hybridizing sequence that is from 17 to 23contiguous nucleobases in length and, optionally, joined at its 5′ endto a promoter sequence.

In some aspects of the combination of at least two amplificationoligomers, the target hybridizing sequence of the first amplificationoligomer is contained within SEQ ID NO:6. In some aspects, the targethybridizing sequence of the first amplification oligomer contains SEQ IDNO:7. In some aspects, the target hybridizing sequence of the firstamplification oligomer contains SEQ ID NO:8. In some aspects, the firstamplification oligomer comprises a target hybridizing sequence that isselected from the group consisting of: SEQ ID NOs:36 to 48. In someaspects, the target hybridizing sequence of the second amplificationoligomer is selected from the group consisting of: SEQ ID NO:123, SEQ IDNO:124, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, & SEQ ID NO:147.In some aspects, the second amplification oligomer is joined at its 5′end to a promoter sequence that is a T7 promoter sequence. In someaspects, the T7 promoter sequence consists of SEQ ID NO:179. In someaspects, the second amplification oligomer comprises a sequence selectedfrom the group consisting of: SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100,SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ IDNO:105, SEQ ID NO:106, SEQ ID NO:109, and SEQ ID NO:110.

In some aspects of the combination of at least two amplificationoligomers, the combination of first amplification oligomer and secondamplification oligomer comprise nucleic acid sequences selected from oneof the following groups: (i) SEQ ID NO:36 and SEQ ID NO:103; (ii) SEQ IDNO:36 and SEQ ID NO:104; (iii) SEQ ID NO:36 and SEQ ID NO:105; (iv) SEQID NO:37 and SEQ ID NO:102; (v) SEQ ID NO:37 and SEQ ID NO:103; (vi) SEQID NO:37 and SEQ ID NO:105; (vii) SEQ ID NO:38 and SEQ ID NO:105; (viii)SEQ ID NO:39 and SEQ ID NO:104; (ix) SEQ ID NO:39 and SEQ ID NO:105; (x)SEQ ID NO:39 and SEQ ID NO:106; (xi) SEQ ID NO:41 and SEQ ID NO:104;(xii) SEQ ID NO:41 and SEQ ID NO:106; (xiii) SEQ ID NO:43 and SEQ IDNO:106; (xiv) SEQ ID NO:45 and SEQ ID NO:103; (xv) SEQ ID NO:45 and SEQID NO:105; (xvi) SEQ ID NO:45 and SEQ ID NO:106; (xvii) SEQ ID NO:46 andSEQ ID NO:106; (xviii) SEQ ID NO:47 and SEQ ID NO:104; and (xix) SEQ IDNO:47 and SEQ ID NO:106.

Further disclosed herein is a detection probe oligomer for the detectionof a Zika virus nucleic acid, wherein the detection probe oligomercomprises: (i) a target hybridizing sequence configured to hybridizeunder stringent conditions to a target sequence of a Zika virus nucleicacid, and, optionally, one or more nucleobases that are notcomplementary to the Zika virus target nucleic acid, and (ii) adetectable label, wherein the Zika virus target nucleic acid sequence isselected from the group consisting of: SEQ ID NO:187, SEQ ID NO:188, &SEQ ID NO:189, including a complement thereof, and/or an RNA equivalentthereof. In some aspects, the detection probe oligomer comprises atarget hybridizing sequence that is configured to selectively hybridizea sequence selected from the group consisting of: SEQ ID NO:194, SEQ IDNO:195, SEQ ID NO:196, SEQ ID NO:197, & SEQ ID NO:198, including acomplement thereof, and/or an RNA equivalent thereof. In some aspects,the target hybridizing sequence of the detection probe oligomer is from17 to 23 contiguous nucleobases in length. In some aspects, the targethybridizing sequence of the detection probe oligomer is selected fromthe group consisting of: SEQ ID NOs:54 to 63 and 66 to 73. In someaspects, the target hybridizing sequence of the detection probe oligomercomprises the one or more nucleobases that are not complementary to thetarget nucleic acid. In some aspects, the target hybridizing sequence ofthe detection probe oligomer comprises from 3 to 7 contiguousnucleobases that are not complementary to the target nucleic acid andthat are joined to one of the 3′ end or the 5′ end of the targethybridizing sequence. In some aspects, the 3 to 7 contiguous nucleobasesthat are not complementary to the target nucleic acid, are complementaryto a portion of the target hybridizing sequence of the detection probeoligomer. In some aspects, the detection probe oligomer comprises adetectable label that is a fluorescent label, a luminescent label, achromophore label, a radionuclide label, a ligand label, an enzymelabel, or a reactive group label. In some aspects, the detectable labelis a chemiluminescent label. In some aspects, the detectable label is anacridinium ester. In some aspects, the detectable label is a fluorescentlabel and the detection probe oligomer further comprises a quenchingcompound. In some aspects, the detection probe oligomer is selected fromthe group consisting of TaqMan detection probes, molecular beacons, andmolecular torches. In some aspects, the target hybridizing sequence ofthe detection probe oligomer does not comprise the one or morenucleobases that are not complementary to the target nucleic acid.

Further disclosed herein is an amplification reaction mixture comprisinga buffered aqueous solution comprising any of the above describedcombinations of at least two amplification oligomers. Further disclosedherein is an amplification reaction mixture, comprising a driedcomposition comprising any of the above described combinations of atleast two amplification oligomers. In some aspects, the driedcomposition comprises a bulking agent. In some aspects, the driedcomposition comprises less than 5% (w/w) of a bulking agent. In someaspects, the bulking agent is a disaccharide form of an amorphous sugar.In some aspects, the bulking agent is one or more of mannitol, trehalosesucrose, lactose, sorbitol, raffinose, and glucose. In some aspects, thedried composition further comprises at least one polymerase enzyme.

Further disclosed herein is a multiplex amplification reaction mixture,wherein the mixture is a buffered aqueous solution comprising at leasttwo combinations of at least two amplification oligomers selected fromthe group consisting of: (A) at least two amplification oligomers foramplifying a target sequence of a Zika virus target nucleic acid,wherein the target sequence consists essentially of SEQ ID NO:189,accounting for complements thereof, RNA equivalents thereof, or both,wherein the first amplification oligomer and the second amplificationoligomer are configured to hybridize to opposite ends of the targetsequence, to generate amplification products; wherein a firstamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 26 contiguous nucleobases in length, and wherein a secondamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 26 contiguous nucleobases in length and, optionally, joinedat its 5′ end to a promoter sequence, combined with at least twoamplification oligomers for amplifying a Zika virus nucleic acid in asample, the oligomer combination comprising: at least two amplificationoligomers for amplifying a target sequence of a Zika virus targetnucleic acid, wherein the target sequence consists essentially of SEQ IDNO:187, accounting for complements thereof, RNA equivalents thereof, orboth, wherein the first amplification oligomer and the secondamplification oligomer are configured to hybridize to opposite ends ofthe target sequence, including complements thereof and/or RNAequivalents thereof, to generate amplification products; wherein a firstamplification oligomer comprises a target hybridizing sequence that isfrom 19 to 23 contiguous nucleobases in length, and wherein a secondamplification oligomer comprises a target hybridizing sequence that isfrom 19 to 25 contiguous nucleobases in length and, optionally, joinedat its 5′ end to a promoter sequence; (B) at least two amplificationoligomers for amplifying a target sequence of a Zika virus targetnucleic acid, wherein the target sequence consists essentially of SEQ IDNO:189, accounting for complements thereof, RNA equivalents thereof, orboth, wherein the first amplification oligomer and the secondamplification oligomer are configured to hybridize to opposite ends ofthe target sequence, to generate amplification products; wherein a firstamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 26 contiguous nucleobases in length, and wherein a secondamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 26 contiguous nucleobases in length and, optionally, joinedat its 5′ end to a promoter sequence, combined with at least twoamplification oligomers for amplifying a Zika virus nucleic acid in asample, the oligomer combination comprising: at least two amplificationoligomers for amplifying a target sequence of a Zika virus targetnucleic acid, wherein the target sequence consists essentially of SEQ IDNO:188, accounting for complements thereof, RNA equivalents thereof, orboth, wherein the first amplification oligomer and the secondamplification oligomer are configured to hybridize to opposite ends ofthe target sequence, including complements thereof and/or RNAequivalents thereof, to generate amplification products; wherein a firstamplification oligomer comprises a target hybridizing sequence that isfrom 19 to 23 contiguous nucleobases in length, and wherein a secondamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 23 contiguous nucleobases in length and, optionally, joinedat its 5′ end to a promoter sequence; (C) at least two amplificationoligomers for amplifying a Zika virus nucleic acid in a sample, theoligomer combination comprising: at least two amplification oligomersfor amplifying a target sequence of a Zika virus target nucleic acid,wherein the target sequence consists essentially of SEQ ID NO:187,accounting for complements thereof, RNA equivalents thereof, or both,wherein the first amplification oligomer and the second amplificationoligomer are configured to hybridize to opposite ends of the targetsequence, including complements thereof and/or RNA equivalents thereof,to generate amplification products; wherein a first amplificationoligomer comprises a target hybridizing sequence that is from 19 to 23contiguous nucleobases in length, and wherein a second amplificationoligomer comprises a target hybridizing sequence that is from 19 to 25contiguous nucleobases in length and, optionally, joined at its 5′ endto a promoter sequence, combined with at least two amplificationoligomers for amplifying a Zika virus nucleic acid in a sample, theoligomer combination comprising: at least two amplification oligomersfor amplifying a target sequence of a Zika virus target nucleic acid,wherein the target sequence consists essentially of SEQ ID NO:188,accounting for complements thereof, RNA equivalents thereof, or both,wherein the first amplification oligomer and the second amplificationoligomer are configured to hybridize to opposite ends of the targetsequence, including complements thereof and/or RNA equivalents thereof,to generate amplification products; wherein a first amplificationoligomer comprises a target hybridizing sequence that is from 19 to 23contiguous nucleobases in length, and wherein a second amplificationoligomer comprises a target hybridizing sequence that is from 17 to 23contiguous nucleobases in length and, optionally, joined at its 5′ endto a promoter sequence; and (D) at least two amplification oligomers foramplifying a target sequence of a Zika virus target nucleic acid,wherein the target sequence consists essentially of SEQ ID NO:189,accounting for complements thereof, RNA equivalents thereof, or both,wherein the first amplification oligomer and the second amplificationoligomer are configured to hybridize to opposite ends of the targetsequence, to generate amplification products; wherein a firstamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 26 contiguous nucleobases in length, and wherein a secondamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 26 contiguous nucleobases in length and, optionally, joinedat its 5′ end to a promoter sequence, combined with at least twoamplification oligomers for amplifying a Zika virus nucleic acid in asample, the oligomer combination comprising: at least two amplificationoligomers for amplifying a target sequence of a Zika virus targetnucleic acid, wherein the target sequence consists essentially of SEQ IDNO:187, accounting for complements thereof, RNA equivalents thereof, orboth, wherein the first amplification oligomer and the secondamplification oligomer are configured to hybridize to opposite ends ofthe target sequence, including complements thereof and/or RNAequivalents thereof, to generate amplification products; wherein a firstamplification oligomer comprises a target hybridizing sequence that isfrom 19 to 23 contiguous nucleobases in length, and wherein a secondamplification oligomer comprises a target hybridizing sequence that isfrom 19 to 25 contiguous nucleobases in length and, optionally, joinedat its 5′ end to a promoter sequence, and combined with at least twoamplification oligomers for amplifying a Zika virus nucleic acid in asample, the oligomer combination comprising: at least two amplificationoligomers for amplifying a target sequence of a Zika virus targetnucleic acid, wherein the target sequence consists essentially of SEQ IDNO:188, accounting for complements thereof, RNA equivalents thereof, orboth, wherein the first amplification oligomer and the secondamplification oligomer are configured to hybridize to opposite ends ofthe target sequence, including complements thereof and/or RNAequivalents thereof, to generate amplification products; wherein a firstamplification oligomer comprises a target hybridizing sequence that isfrom 19 to 23 contiguous nucleobases in length, and wherein a secondamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 23 contiguous nucleobases in length and, optionally, joinedat its 5′ end to a promoter sequence.

Further disclosed herein is a multiplex amplification reaction mixture,wherein the mixture is a dried composition comprising at least twocombinations of at least two amplification oligomers selected from thegroup consisting of: (A) at least two amplification oligomers foramplifying a target sequence of a Zika virus target nucleic acid,wherein the target sequence consists essentially of SEQ ID NO:189,accounting for complements thereof, RNA equivalents thereof, or both,wherein the first amplification oligomer and the second amplificationoligomer are configured to hybridize to opposite ends of the targetsequence, to generate amplification products; wherein a firstamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 26 contiguous nucleobases in length, and wherein a secondamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 26 contiguous nucleobases in length and, optionally, joinedat its 5′ end to a promoter sequence, combined with at least twoamplification oligomers for amplifying a Zika virus nucleic acid in asample, the oligomer combination comprising: at least two amplificationoligomers for amplifying a target sequence of a Zika virus targetnucleic acid, wherein the target sequence consists essentially of SEQ IDNO:187, accounting for complements thereof, RNA equivalents thereof, orboth, wherein the first amplification oligomer and the secondamplification oligomer are configured to hybridize to opposite ends ofthe target sequence, including complements thereof and/or RNAequivalents thereof, to generate amplification products; wherein a firstamplification oligomer comprises a target hybridizing sequence that isfrom 19 to 23 contiguous nucleobases in length, and wherein a secondamplification oligomer comprises a target hybridizing sequence that isfrom 19 to 25 contiguous nucleobases in length and, optionally, joinedat its 5′ end to a promoter sequence; (B) at least two amplificationoligomers for amplifying a target sequence of a Zika virus targetnucleic acid, wherein the target sequence consists essentially of SEQ IDNO:189, accounting for complements thereof, RNA equivalents thereof, orboth, wherein the first amplification oligomer and the secondamplification oligomer are configured to hybridize to opposite ends ofthe target sequence, to generate amplification products; wherein a firstamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 26 contiguous nucleobases in length, and wherein a secondamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 26 contiguous nucleobases in length and, optionally, joinedat its 5′ end to a promoter sequence, combined with at least twoamplification oligomers for amplifying a Zika virus nucleic acid in asample, the oligomer combination comprising: at least two amplificationoligomers for amplifying a target sequence of a Zika virus targetnucleic acid, wherein the target sequence consists essentially of SEQ IDNO:188, accounting for complements thereof, RNA equivalents thereof, orboth, wherein the first amplification oligomer and the secondamplification oligomer are configured to hybridize to opposite ends ofthe target sequence, including complements thereof and/or RNAequivalents thereof, to generate amplification products; wherein a firstamplification oligomer comprises a target hybridizing sequence that isfrom 19 to 23 contiguous nucleobases in length, and wherein a secondamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 23 contiguous nucleobases in length and, optionally, joinedat its 5′ end to a promoter sequence; (C) at least two amplificationoligomers for amplifying a Zika virus nucleic acid in a sample, theoligomer combination comprising: at least two amplification oligomersfor amplifying a target sequence of a Zika virus target nucleic acid,wherein the target sequence consists essentially of SEQ ID NO:187,accounting for complements thereof, RNA equivalents thereof, or both,wherein the first amplification oligomer and the second amplificationoligomer are configured to hybridize to opposite ends of the targetsequence, including complements thereof and/or RNA equivalents thereof,to generate amplification products; wherein a first amplificationoligomer comprises a target hybridizing sequence that is from 19 to 23contiguous nucleobases in length, and wherein a second amplificationoligomer comprises a target hybridizing sequence that is from 19 to 25contiguous nucleobases in length and, optionally, joined at its 5′ endto a promoter sequence, combined with at least two amplificationoligomers for amplifying a Zika virus nucleic acid in a sample, theoligomer combination comprising: at least two amplification oligomersfor amplifying a target sequence of a Zika virus target nucleic acid,wherein the target sequence consists essentially of SEQ ID NO:188,accounting for complements thereof, RNA equivalents thereof, or both,wherein the first amplification oligomer and the second amplificationoligomer are configured to hybridize to opposite ends of the targetsequence, including complements thereof and/or RNA equivalents thereof,to generate amplification products; wherein a first amplificationoligomer comprises a target hybridizing sequence that is from 19 to 23contiguous nucleobases in length, and wherein a second amplificationoligomer comprises a target hybridizing sequence that is from 17 to 23contiguous nucleobases in length and, optionally, joined at its 5′ endto a promoter sequence; and (D) at least two amplification oligomers foramplifying a target sequence of a Zika virus target nucleic acid,wherein the target sequence consists essentially of SEQ ID NO:189,accounting for complements thereof, RNA equivalents thereof, or both,wherein the first amplification oligomer and the second amplificationoligomer are configured to hybridize to opposite ends of the targetsequence, to generate amplification products; wherein a firstamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 26 contiguous nucleobases in length, and wherein a secondamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 26 contiguous nucleobases in length and, optionally, joinedat its 5′ end to a promoter sequence, combined with at least twoamplification oligomers for amplifying a Zika virus nucleic acid in asample, the oligomer combination comprising: at least two amplificationoligomers for amplifying a target sequence of a Zika virus targetnucleic acid, wherein the target sequence consists essentially of SEQ IDNO:187, accounting for complements thereof, RNA equivalents thereof, orboth, wherein the first amplification oligomer and the secondamplification oligomer are configured to hybridize to opposite ends ofthe target sequence, including complements thereof and/or RNAequivalents thereof, to generate amplification products; wherein a firstamplification oligomer comprises a target hybridizing sequence that isfrom 19 to 23 contiguous nucleobases in length, and wherein a secondamplification oligomer comprises a target hybridizing sequence that isfrom 19 to 25 contiguous nucleobases in length and, optionally, joinedat its 5′ end to a promoter sequence, and combined with at least twoamplification oligomers for amplifying a Zika virus nucleic acid in asample, the oligomer combination comprising: at least two amplificationoligomers for amplifying a target sequence of a Zika virus targetnucleic acid, wherein the target sequence consists essentially of SEQ IDNO:188, accounting for complements thereof, RNA equivalents thereof, orboth, wherein the first amplification oligomer and the secondamplification oligomer are configured to hybridize to opposite ends ofthe target sequence, including complements thereof and/or RNAequivalents thereof, to generate amplification products; wherein a firstamplification oligomer comprises a target hybridizing sequence that isfrom 19 to 23 contiguous nucleobases in length, and wherein a secondamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 23 contiguous nucleobases in length and, optionally, joinedat its 5′ end to a promoter sequence. In some aspects, the driedcomposition comprises a bulking agent. In some aspects, the driedcomposition comprises less than 5% (w/w) of a bulking agent. In someaspects, the bulking agent is a disaccharide form of an amorphous sugar.In some aspects, the bulking agent is one or more of mannitol, trehalosesucrose, lactose, sorbitol, raffinose, and glucose. In some aspects, thedried composition further comprises at least one polymerase enzyme.

Further disclosed herein is a kit comprising any of the at least twoamplification oligomers described herein. In some aspects, the firstamplification oligomer is in a first container within the kit and thesecond amplification oligomer is in a second container within the kit.In one aspect, the kit further comprises a detection probe oligomer. Inone aspect, the detection probe oligomer is any detection probe oligomerdescribed herein.

Further disclosed herein is a detection reaction mixture comprising abuffered aqueous solution and a detection probe oligomer for thedetection of a Zika virus nucleic acid, wherein the detection probeoligomer comprises: (i) a target hybridizing sequence configured tohybridize under stringent conditions to a target sequence of a Zikavirus nucleic acid, and, optionally, one or more nucleobases that arenot complementary to the Zika virus target nucleic acid, and (ii) adetectable label, wherein the Zika virus target nucleic acid sequence isselected from the group consisting of: SEQ ID NO:187, SEQ ID NO:188, &SEQ ID NO:189, including a complement thereof, and/or an RNA equivalentthereof. In some aspects, the detection probe oligomer comprises atarget hybridizing sequence that is configured to selectively hybridizea sequence selected from the group consisting of: SEQ ID NO:194, SEQ IDNO:195, SEQ ID NO:196, SEQ ID NO:197, & SEQ ID NO:198, including acomplement thereof, and/or an RNA equivalent thereof. In some aspects,the target hybridizing sequence of the detection probe oligomer is from17 to 23 contiguous nucleobases in length. In some aspects, the targethybridizing sequence of the detection probe oligomer is selected fromthe group consisting of: SEQ ID NOs:54 to 63 and 66 to 73. In someaspects, the target hybridizing sequence of the detection probe oligomercomprises the one or more nucleobases that are not complementary to thetarget nucleic acid. In some aspects, the target hybridizing sequence ofthe detection probe oligomer comprises from 3 to 7 contiguousnucleobases that are not complementary to the target nucleic acid andthat are joined to one of the 3′ end or the 5′ end of the targethybridizing sequence. In some aspects, the 3 to 7 contiguous nucleobasesthat are not complementary to the target nucleic acid, are complementaryto a portion of the target hybridizing sequence of the detection probeoligomer. In some aspects, the detection probe oligomer comprises adetectable label that is a fluorescent label, a luminescent label, achromophore label, a radionuclide label, a ligand label, an enzymelabel, or a reactive group label. In some aspects, the detectable labelis a chemiluminescent label. In some aspects, the detectable label is anacridinium ester. In some aspects, the detectable label is a fluorescentlabel and the detection probe oligomer further comprises a quenchingcompound. In some aspects, the detection probe oligomer is selected fromthe group consisting of TaqMan detection probes, molecular beacons, andmolecular torches. In some aspects, the target hybridizing sequence ofthe detection probe oligomer does not comprise the one or morenucleobases that are not complementary to the target nucleic acid.

Further disclosed herein is a dried composition comprising a detectionprobe oligomer for the detection of a Zika virus nucleic acid, whereinthe detection probe oligomer comprises: (i) a target hybridizingsequence configured to hybridize under stringent conditions to a targetsequence of a Zika virus nucleic acid, and, optionally, one or morenucleobases that are not complementary to the Zika virus target nucleicacid, and (ii) a detectable label, wherein the Zika virus target nucleicacid sequence is selected from the group consisting of: SEQ ID NO:187,SEQ ID NO:188, & SEQ ID NO:189, including a complement thereof, and/oran RNA equivalent thereof. In some aspects, the detection probe oligomercomprises a target hybridizing sequence that is configured toselectively hybridize a sequence selected from the group consisting of:SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, & SEQ IDNO:198, including a complement thereof, and/or an RNA equivalentthereof. In some aspects, the target hybridizing sequence of thedetection probe oligomer is from 17 to 23 contiguous nucleobases inlength. In some aspects, the target hybridizing sequence of thedetection probe oligomer is selected from the group consisting of: SEQID NOs:54 to 63 and 66 to 73. In some aspects, the target hybridizingsequence of the detection probe oligomer comprises the one or morenucleobases that are not complementary to the target nucleic acid. Insome aspects, the target hybridizing sequence of the detection probeoligomer comprises from 3 to 7 contiguous nucleobases that are notcomplementary to the target nucleic acid and that are joined to one ofthe 3′ end or the 5′ end of the target hybridizing sequence. In someaspects, the 3 to 7 contiguous nucleobases that are not complementary tothe target nucleic acid, are complementary to a portion of the targethybridizing sequence of the detection probe oligomer. In some aspects,the detection probe oligomer comprises a detectable label that is afluorescent label, a luminescent label, a chromophore label, aradionuclide label, a ligand label, an enzyme label, or a reactive grouplabel. In some aspects, the detectable label is a chemiluminescentlabel. In some aspects, the detectable label is an acridinium ester. Insome aspects, the detectable label is a fluorescent label and thedetection probe oligomer further comprises a quenching compound. In someaspects, the detection probe oligomer is selected from the groupconsisting of TaqMan detection probes, molecular beacons, and moleculartorches. In some aspects, the target hybridizing sequence of thedetection probe oligomer does not comprise the one or more nucleobasesthat are not complementary to the target nucleic acid. In some aspects,the dried composition comprises a bulking agent. In some aspects, thedried composition comprises less than 5% (w/w) of a bulking agent. Insome aspects, the bulking agent is a disaccharide form of an amorphoussugar. In some aspects, the bulking agent is one or more of mannitol,trehalose sucrose, lactose, sorbitol, raffinose, and glucose. In someaspects, the dried composition further comprises at least one polymeraseenzyme. In some aspects, the dried composition further comprises one ormore of the combinations of at least two amplification oligomersdescribed herein.

Further disclosed herein are methods for determining the presence orabsence of a Zika virus nucleic acid in a sample, the method comprisingthe steps of: (A) contacting a sample with a combination of at least twoamplification oligomers for amplifying a target sequence of a Zika virustarget nucleic acid, wherein the target sequence consists essentially ofSEQ ID NO:189, accounting for complements thereof, RNA equivalentsthereof, or both, wherein the first amplification oligomer and thesecond amplification oligomer are configured to hybridize to oppositeends of the target sequence to generate amplification products; whereina first amplification oligomer comprises a target hybridizing sequencethat is from 17 to 26 contiguous nucleobases in length, and wherein asecond amplification oligomer comprises a target hybridizing sequencethat is from 17 to 26 contiguous nucleobases in length and, optionally,joined at its 5′ end to a promoter sequence; (B) performing an in vitronucleic acid amplification reaction, wherein any Zika virus targetsequence present in the sample is used as a template for generating anamplification product; and (C) detecting the presence or absence of theamplification product, thereby determining the presence or absence ofthe Zika virus nucleic acid in the sample. In some aspects, the targethybridizing sequence of the first amplification oligomer is containedwithin SEQ ID NO:24 and contains SEQ ID NO:25. In some aspects, thefirst amplification oligomer comprises a sequence that is selected fromthe group consisting of: SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ IDNO:52, and SEQ ID NO:53. In some aspects, the target hybridizing regionof the second amplification oligomer is contained within SEQ ID NO:150.In some aspects, the target hybridizing region of the secondamplification oligomer is 19 contiguous nucleobases in length or is 20contiguous nucleobases in length. In some aspects, the targethybridizing sequence of the second amplification oligomer is selectedfrom the group consisting of: SEQ ID NO:113, SEQ ID NO:119, SEQ IDNO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:130, and SEQ ID NO:131.In some aspects, the target hybridizing sequence of the secondamplification oligomer is selected from the group consisting of: SEQ IDNO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:121, SEQID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129,SEQ ID NO:130, SEQ ID NO:131, and SEQ ID NO:134. In some aspects, thesecond amplification oligomer is joined at its 5′ end to a promotersequence that is a T7 promoter sequence. In some aspects, the T7promoter sequence consists of SEQ ID NO:179. In some aspects, the secondamplification oligomer comprises a sequences selected from the groupconsisting of: SEQ ID NOs:74 to 89, SEQ ID NO:107, and SEQ ID NO:108. Insome aspects, the combination of first amplification oligomer and secondamplification oligomer comprise nucleic acid sequences selected from thegroup consisting of: (i) SEQ ID NO:16 and SEQ ID NO:78; (ii) SEQ IDNO:16 and SEQ ID NO:80; (iii) SEQ ID NO:16 and SEQ ID NO:75; (iv) SEQID NO:16 and SEQ ID NO:89; (v) SEQ ID NO:16 and SEQ ID NO:74; (vi) SEQID NO:11 and SEQ ID NO:78; (vii) SEQ ID NO:11 and SEQ ID NO:80; (viii)SEQ ID NO:11 and SEQ ID NO:75; (ix) SEQ ID NO:11 and SEQ ID NO:89; (x)SEQ ID NO:11 and SEQ ID NO:74; (xi) SEQ ID NO:12 and SEQ ID NO:79; (xii)SEQ ID NO:12 and SEQ ID NO:87; (xiii) SEQ ID NO:12 and SEQ ID NO:89;(xiv) SEQ ID NO:12 and SEQ ID NO:74; (xv) SEQ ID NO:17 and SEQ ID NO:79;(xvi) SEQ ID NO:17 and SEQ ID NO:87; (xvii) SEQ ID NO:17 and SEQ IDNO:89; (xviii) SEQ ID NO:17 and SEQ ID NO:74; (xix) SEQ ID NO:53 and SEQID NO:78; (xx) SEQ I DNO:53 and SEQ ID NO:80; (xxi) SEQ ID NO:53 and SEQID NO:75; (xxii) SEQ ID NO:53 and SEQ ID NO:89; (xxiii) SEQ ID NO:53 andSEQ ID NO:74; (xxiv) SEQ ID NO:18 and SEQ ID NO:78; (xxv) SEQ I DNO:18and SEQ ID NO:80; (xxvi) SEQ ID NO:18 and SEQ ID NO:75; (xxvii) SEQ IDNO:18 and SEQ ID NO:89; and (xxviii) SEQ ID NO:18 and SEQ ID NO:74. Inone aspect, the combination of at least two amplification oligomerscomprises a third amplification oligomer for amplifying a targetsequence of a Zika virus nucleic acid, wherein the third amplificationoligomer comprises a target hybridizing sequence selected from the groupconsisting of: SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:26,SEQ ID NO:27, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52,and SEQ ID NO:53, and wherein the target hybridizing sequence of thefirst amplification oligomer and the target hybridizing sequence of thethird amplification oligomer are different sequences. In one aspect, thecombination of first amplification oligomer, second amplificationoligomer and third amplification oligomer is: (i) a first amplificationoligomer comprising a nucleic acid sequence consisting of SEQ ID NO:16;a second amplification oligomer comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NOs:74, 75, 78, 80, & 89;and a third amplification oligomer comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NOs:11, 12, 17, 18, & 53;(ii) a first amplification oligomer comprising a nucleic acid sequenceconsisting of SEQ ID NO:11; a second amplification oligomer comprising anucleic acid sequence selected from the group consisting of SEQ IDNOs:74, 75, 78, 80, & 89; and a third amplification oligomer comprisinga nucleic acid sequence selected from the group consisting of SEQ IDNOs:12, 17, 18, & 53; (iii) a first amplification oligomer comprising anucleic acid sequence consisting of SEQ ID NO:12; a second amplificationoligomer comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs:74, 75, 78, 80, & 89; and a third amplificationoligomer comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs:17, 18, & 53; (iv) a first amplificationoligomer comprising a nucleic acid sequence consisting of SEQ ID NO:17;a second amplification oligomer comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NOs:74, 75, 78, 80, & 89;and a third amplification oligomer comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NOs:18, & 53; or (v) afirst amplification oligomer comprising a nucleic acid sequenceconsisting of SEQ ID NO:18; a second amplification oligomer comprising anucleic acid sequence selected from the group consisting of SEQ IDNOs:74, 75, 78, 80, & 89; and a third amplification oligomer comprisinga nucleic acid sequence consisting of SEQ ID NO:53. In some aspects, thecombination of at least two amplification oligomers comprises a fourthamplification oligomer for amplifying a target sequence of a Zikanucleic acid, wherein the fourth amplification oligomer comprises atarget hybridizing sequence selected from the group consisting of: SEQID NO113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:125, SEQ ID NO:127,SEQ ID NO:128, & SEQ ID NO:134, and wherein the target hybridizingsequence of the fourth amplification oligomer is optionally joined atits 5′ end to a promoter sequence, and wherein the target hybridizingsequence of the second amplification oligomer and the target hybridizingsequence of the fourth amplification oligomer are different sequences.In some aspects, the target hybridizing sequence of the fourthamplification oligomer is joined to a promoter sequence is a T7 promotersequence, wherein preferably the promoter sequence consists of SEQ IDNO:179. In some aspects, the fourth amplification oligomer comprises asequence that is selected from the group consisting of: SEQ ID NO:74,SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:87,and SEQ ID NO:89. In some aspects, the detecting step comprisescontacting the in vitro amplification reaction with at least onedetection probe oligomer, wherein the detection probe oligomercomprises: (i) a target hybridizing sequence and, optionally, one ormore nucleobases that are not complementary to the Zika virus targetnucleic acid, and (ii) a detectable label. In some aspects, the targethybridizing sequence of the detection probe oligomer is from 17 to 23contiguous nucleobases in length. In some aspects, the targethybridizing sequence of the detection probe oligomer is selected fromthe group consisting of: SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NOs:66to 73. In some aspects, the target hybridizing sequence of the detectionprobe oligomer comprises the one or more nucleobases that are notcomplementary to the target nucleic acid. In some aspects, the targethybridizing sequence of the detection probe oligomer comprises at eitherits 3′ end or its 5′ end from 3 to 7 contiguous nucleobases that are notcomplementary to the target sequence of the Zika virus nucleic acid. Insome aspects, the 3 to 7 contiguous nucleobases that are notcomplementary to the target sequence, are complementary to a portion ofthe target hybridizing sequence of the detection probe oligomer. In someaspects, the detectable label is a fluorescent label, a luminescentlabel, a chromophore label, a radionuclide label, a ligand label, anenzyme label, or a reactive group label. In some aspects, the detectablelabel is a chemiluminescent label. In some aspects, the detectable labelis an acridinium ester. In some aspects, the detectable label is afluorescent label and wherein the detection probe oligomer furthercomprises a quenching compound. In some aspects, the detection probeoligomer is selected from the group consisting of TaqMan detectionprobes, molecular beacons, and molecular torches. In some aspects, thetarget hybridizing sequence does not comprise the one or morenucleobases that are not complementary to the target nucleic acid. Insome aspects, the combination of the first amplification oligomer andthe second amplification oligomer is selected from the group consistingof: (i) SEQ ID NO:16 and SEQ ID NO:78; (ii) SEQ I DNO:16 and SEQ IDNO:80; (iii) SEQ ID NO:16 and SEQ ID NO:75; (iv) SEQ ID NO:16 and SEQ IDNO:89; (v) SEQ ID NO:16 and SEQ ID NO:74; (vi) SEQ ID NO:11 and SEQ IDNO:78; (vii) SEQ ID NO:11 and SEQ ID NO:80; (viii) SEQ ID NO:11 and SEQID NO:75; (ix) SEQ ID NO:11 and SEQ ID NO:89; (x) SEQ ID NO:11 and SEQID NO:74; (xi) SEQ ID NO:12 and SEQ ID NO:79; (xii) SEQ ID NO:12 and SEQID NO:87; (xiii) SEQ ID NO:12 and SEQ ID NO:89; (xiv) SEQ ID NO:12 andSEQ ID NO:74; (xv) SEQ ID NO:17 and SEQ ID NO:79; (xvi) SEQ ID NO:17 andSEQ ID NO:87; (xvii) SEQ ID NO:17 and SEQ ID NO:89; (xviii) SEQ ID NO:17and SEQ ID NO:74; (xix) SEQ ID NO:53 and SEQ ID NO:78; (xx) SEQ I DNO:53and SEQ ID NO:80; (xxi) SEQ ID NO:53 and SEQ ID NO:75; (xxii) SEQ IDNO:53 and SEQ ID NO:89; (xxiii) SEQ ID NO:53 and SEQ ID NO:74; (xxiv)SEQ ID NO:18 and SEQ ID NO:78; (xxv) SEQ I DNO:18 and SEQ ID NO:80;(xxvi) SEQ ID NO:18 and SEQ ID NO:75; (xxvii) SEQ ID NO:18 and SEQ IDNO:89; and (xxviii) SEQ ID NO:18 and SEQ ID NO:74, and wherein thedetection probe oligomer comprises a target hybridizing sequenceconsisting of SEQ ID NO:69, and the detectable label consists of anacridinium ester label. In some aspects, the combination comprises athird amplification oligomer for amplifying a target sequence of a Zikavirus nucleic acid, wherein the third amplification oligomer comprises atarget hybridizing sequence selected from the group consisting of: SEQID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, andwherein the target hybridizing sequence of the first amplificationoligomer and the target hybridizing sequence of the third amplificationoligomer are different sequences. In some aspects, the combination offirst amplification oligomer, second amplification oligomer and thirdamplification oligomer is: (i) a first amplification oligomer comprisinga nucleic acid sequence consisting of SEQ ID NO:16; a secondamplification oligomer comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs:74, 75, 78, 80, & 89; and a thirdamplification oligomer comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs:11, 12, 17, 18, & 53; (ii) a firstamplification oligomer comprising a nucleic acid sequence consisting ofSEQ ID NO:11; a second amplification oligomer comprising a nucleic acidsequence selected from the group consisting of SEQ ID NOs:74, 75, 78,80, & 89; and a third amplification oligomer comprising a nucleic acidsequence selected from the group consisting of SEQ ID NOs:12, 17, 18, &53; (iii) a first amplification oligomer comprising a nucleic acidsequence consisting of SEQ ID NO:12; a second amplification oligomercomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NOs:74, 75, 78, 80, & 89; and a third amplification oligomercomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NOs:17, 18, & 53; (iv) a first amplification oligomer comprisinga nucleic acid sequence consisting of SEQ ID NO:17; a secondamplification oligomer comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs:74, 75, 78, 80, & 89; and a thirdamplification oligomer comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs:18, & 53; or (v) a firstamplification oligomer comprising a nucleic acid sequence consisting ofSEQ ID NO:18; a second amplification oligomer comprising a nucleic acidsequence selected from the group consisting of SEQ ID NOs:74, 75, 78,80, & 89; and a third amplification oligomer comprising a nucleic acidsequence consisting of SEQ ID NO:53. In some aspects, the combinationcomprises a fourth amplification oligomer for amplifying a targetsequence of a Zika nucleic acid, wherein the fourth amplificationoligomer comprises a target hybridizing sequence selected from the groupconsisting of: SEQ ID NO113, SEQ ID NO:114, SEQ ID NO:115, SEQ IDNO:125, SEQ ID NO:127, SEQ ID NO:128, & SEQ ID NO:134, and wherein thetarget hybridizing sequence of the fourth amplification oligomer isoptionally joined at its 5′ end to a promoter sequence, and wherein thetarget hybridizing sequence of the second amplification oligomer and thetarget hybridizing sequence of the fourth amplification oligomer aredifferent sequences. In some aspects, the target hybridizing sequence ofthe fourth amplification oligomer is joined to a promoter sequence is aT7 promoter sequence, preferably wherein the promoter sequence consistsof SEQ ID NO:179. In some aspects, the fourth amplification oligomercomprises a sequence that is selected from the group consisting of: SEQID NO:74, SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ IDNO:87, and SEQ ID NO:89. In some aspects, the amplification step (b) isperformed with the detection step (c). In some aspects, before theamplification step (b), the method further comprises purifying the Zikavirus target sequence away from one or more components of the sample. Insome aspects, the purifying step comprises contacting the sample with atleast one capture probe oligomer comprising a target-hybridizingsequence covalently attached to a moiety that binds to an immobilizedprobe, wherein the target-hybridizing sequence is selected from thegroup consisting of SEQ ID NOs:166 to 178. In some aspects, the moietycomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NO:181 and SEQ ID NO:182. In some aspects, the immobilized probecomprises a nucleic acid sequence consisting of SEQ ID NO:180. In someaspects, the capture probe oligomer comprises a nucleic acid sequenceselected from the group consisting of SEQ ID NOs:153 to 165. In someaspects, the in vitro amplification reaction is an isothermalamplification reaction. In some aspects, the in vitro amplificationreaction is a TMA reaction.

Further disclosed herein are methods for determining the presence orabsence of a Zika virus nucleic acid in a sample, the method comprisingthe steps of: (A) contacting a sample with a combination of at least twoamplification oligomers for amplifying a target sequence of a Zika virustarget nucleic acid, wherein the target sequence consists essentially ofSEQ ID NO:187, accounting for complements thereof, RNA equivalentsthereof, or both, wherein the first amplification oligomer and thesecond amplification oligomer are configured to hybridize to oppositeends of the target sequence to generate amplification products; whereina first amplification oligomer comprises a target hybridizing sequencethat is from 19 to 23 contiguous nucleobases in length, and wherein asecond amplification oligomer comprises a target hybridizing sequencethat is from 19 to 25 contiguous nucleobases in length and, optionally,joined at its 5′ end to a promoter sequence; (B) performing an in vitronucleic acid amplification reaction, wherein any Zika virus targetsequence present in the sample is used as a template for generating anamplification product; and (C) detecting the presence or absence of theamplification product, thereby determining the presence or absence ofthe Zika virus nucleic acid in the sample. In some aspects, the targethybridizing sequence of the first amplification oligomer is containedwithin SEQ ID NO:4. In some aspects, the target hybridizing sequence ofthe first amplification oligomer contains SEQ ID NO:2. In some aspects,the first amplification oligomer comprises a sequence selected from thegroup consisting of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32,SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35. In some aspects, thetarget hybridizing sequence of the second amplification oligomer iscontained within SEQ ID NO:152. In some aspects, the target hybridizingsequence of the second amplification oligomer contains SEQ ID NO:148. Insome aspects, the target hybridizing sequence of the secondamplification oligomer contains SEQ ID NO:149. In some aspects, thesecond amplification oligomer is joined at its 5′ end to a promotersequence that is a T7 promoter sequence. In some aspects, the T7promoter sequence consists of SEQ ID NO:179. In some aspects, the targethybridizing sequence of the second amplification oligomer is selectedfrom the group consisting of: SEQ ID NO:111, SEQ ID NO:116, SEQ IDNO:117, SEQ ID NO:122, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, andSEQ ID NO:138. In some aspects, the second amplification oligomercomprises a sequence selected from the group consisting of: SEQ IDNO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ IDNO:95, SEQ ID NO:96, and SEQ ID NO:97. In some aspects, the combinationof first amplification oligomer and second amplification oligomercomprise nucleic acid sequences selected from the group consisting of:(i) SEQ ID NO:30 and SEQ ID NO:92; (ii) SEQ ID NO:30 and SEQ ID NO:94;(iii) SEQ ID NO:30 and SEQ ID NO:95 (iv) SEQ ID NO:35 and SEQ ID NO:92;(v) SEQ ID NO:35 and SEQ ID NO:94; and (vi) SEQ ID NO:35 and SEQ IDNO:95. In some aspects, the combination further comprises a thirdamplification oligomer for amplifying a target sequence of a Zika virusnucleic acid, wherein the third amplification oligomer comprises atarget hybridizing sequence selected from the group consisting of: SEQID NO:111, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:122, SEQ ID NO:135,SEQ ID NO:136, SEQ ID NO:137, & SEQ ID NO:138, wherein, optionally, thetarget hybridizing sequence of the third amplification oligomer isjoined at it 5′ end to a promoter sequence, and wherein the targethybridizing sequence of the second amplification oligomer and the targethybridizing sequence of the third amplification oligomer are differentsequences. In some aspects, the target hybridizing sequence of the thirdamplification oligomer is joined at its 5′ end to a promoter sequencethat is a T7 promoter sequence, preferably wherein the promoter sequenceconsists of SEQ ID NO:179. In some aspects, the third amplificationoligomer comprises a sequence selected from the group consisting of: SEQID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ IDNO:95, SEQ ID NO:96, and SEQ ID NO:97. In some aspects, the detectingstep comprises contacting the in vitro amplification reaction with atleast one detection probe oligomer, wherein the detection probe oligomercomprises: (i) a target hybridizing sequence and, optionally, one ormore nucleobases that are not complementary to the Zika virus targetnucleic acid, and (ii) a detectable label. In some aspects, the targethybridizing sequence of the detection probe oligomer is from 17 to 23contiguous nucleobases in length. In some aspects, the targethybridizing sequence of the detection probe oligomer is selected fromthe group consisting of: SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:58.In some aspects, the target hybridizing sequence of the detection probeoligomer comprises the one or more nucleobases that are notcomplementary to the target nucleic acid. In some aspects, the targethybridizing sequence of the detection probe oligomer comprises at eitherits 3′ end or its 5′ end from 3 to 7 contiguous nucleobases that are notcomplementary to the target sequence of the Zika virus nucleic acid. Insome aspects, the 3 to 7 contiguous nucleobases that are notcomplementary to the target sequence, are complementary to a portion ofthe target hybridizing sequence of the detection probe oligomer. In someaspects, the detectable label is a fluorescent label, a luminescentlabel, a chromophore label, a radionuclide label, a ligand label, anenzyme label, or a reactive group label. In some aspects, the detectablelabel is a chemiluminescent label. In some aspects, the detectable labelis an acridinium ester. In some aspects, the detectable label is afluorescent label and wherein the detection probe oligomer furthercomprises a quenching compound. In some aspects, the detection probeoligomer is selected from the group consisting of TaqMan detectionprobes, molecular beacons, and molecular torches. In some aspects, thedetection probe oligomer comprises a sequence selected from the groupconsisting of: SEQ ID NO:54, SEQ ID NO:55, & SEQ ID NO:58. In someaspects, the combination of the first amplification oligomer and thesecond amplification oligomer is selected from the group consisting of:(i) SEQ ID NO:30 and SEQ ID NO:92; (ii) SEQ ID NO:30 and SEQ ID NO:94;(iii) SEQ ID NO:30 and SEQ ID NO:95 (iv) SEQ ID NO:35 and SEQ ID NO:92;(v) SEQ ID NO:35 and SEQ ID NO:94; and (vi) SEQ ID NO:35 and SEQ IDNO:95, and wherein the detection probe oligomer comprises a targethybridizing sequence consisting of SEQ ID NO:58, and the detectablelabel consists of an acridinium ester label. In some aspects, thecombination further comprises a third amplification oligomer foramplifying a target sequence of a Zika virus nucleic acid, wherein thethird amplification oligomer comprises a target hybridizing sequenceselected from the group consisting of: SEQ ID NO:111, SEQ ID NO:116, SEQID NO:117, SEQ ID NO:122, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, &SEQ ID NO:138, wherein, optionally, the target hybridizing sequence ofthe third amplification oligomer is joined at it 5′ end to a promotersequence, and wherein the target hybridizing sequence of the secondamplification oligomer and the target hybridizing sequence of the thirdamplification oligomer are different sequences. In some aspects, thetarget hybridizing sequence of the third amplification oligomer isjoined at its 5′ end to a promoter sequence that is a T7 promotersequence, preferably wherein the promoter sequence consists of SEQ IDNO:179. In some aspects, the third amplification oligomer comprises asequence selected from the group consisting of: SEQ ID NO:90, SEQ IDNO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ IDNO:96, and SEQ ID NO:97. In some aspects, the amplification step (b) isperformed with the detection step (c). In some aspects, before theamplification step (b), the method further comprises purifying the Zikavirus target sequence away from one or more components of the sample. Insome aspects, the purifying step comprises contacting the sample with atleast one capture probe oligomer comprising a target-hybridizingsequence covalently attached to a moiety that binds to an immobilizedprobe, wherein the target-hybridizing sequence is selected from thegroup consisting of SEQ ID NOs:166 to 178. In some aspects, the moietycomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NO:181 and SEQ ID NO:182. In some aspects, the immobilized probecomprises a nucleic acid sequence consisting of SEQ ID NO:180. In someaspects, the capture probe oligomer comprises a nucleic acid sequenceselected from the group consisting of SEQ ID NOs:153 to 165. In someaspects, the in vitro amplification reaction is an isothermalamplification reaction. In some aspects, the in vitro amplificationreaction is a TMA reaction.

Further disclosed herein are methods for determining the presence orabsence of a Zika virus nucleic acid in a sample, the method comprisingthe steps of: (A) contacting a sample with a combination of at least twoamplification oligomers for amplifying a target sequence of a Zika virustarget nucleic acid, wherein the target sequence consists essentially ofSEQ ID NO:188, accounting for complements thereof, RNA equivalentsthereof, or both, wherein the first amplification oligomer and thesecond amplification oligomer are configured to hybridize to oppositeends of the target sequence to generate amplification products; whereina first amplification oligomer comprises a target hybridizing sequencethat is from 19 to 23 contiguous nucleobases in length, and wherein asecond amplification oligomer comprises a target hybridizing sequencethat is from 17 to 23 contiguous nucleobases in length and, optionally,joined at its 5′ end to a promoter sequence; (B) performing an in vitronucleic acid amplification reaction, wherein any Zika virus targetsequence present in the sample is used as a template for generating anamplification product; and (C) detecting the presence or absence of theamplification product, thereby determining the presence or absence ofthe Zika virus nucleic acid in the sample. In some aspects, the targethybridizing sequence of the first amplification oligomer is containedwithin SEQ ID NO:6. In some aspects, the target hybridizing sequence ofthe first amplification oligomer contains SEQ ID NO:7. In some aspects,the target hybridizing sequence of the first amplification oligomercontains SEQ ID NO:8. In some aspects, the first amplification oligomercomprises a target hybridizing sequence selected from the groupconsisting of: SEQ ID NOs:36 to 48. In some aspects, the targethybridizing sequence of the second amplification oligomer is selectedfrom the group consisting of: SEQ ID NO:123, SEQ ID NO:124, SEQ IDNO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQID NO:144, SEQ ID NO:145, SEQ ID NO:146, & SEQ ID NO:147. In someaspects, the second amplification oligomer is joined at its 5′ end to apromoter sequence that is a T7 promoter sequence. In some aspects, theT7 promoter sequence consists of SEQ ID NO:179. In some aspects, thesecond amplification oligomer comprises a sequence selected from thegroup consisting of: SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ IDNO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQID NO:106, SEQ ID NO:109, and SEQ ID NO:110. In some aspects, thecombination of first amplification oligomer and second amplificationoligomer comprise nucleic acid sequences selected from the groupconsisting of: (i) SEQ ID NO:36 and SEQ ID NO:103; (ii) SEQ ID NO:36 andSEQ ID NO:104; (iii) SEQ ID NO:36 and SEQ ID NO:105; (iv) SEQ ID NO:37and SEQ ID NO:102; (v) SEQ ID NO:37 and SEQ ID NO:103; (vi) SEQ ID NO:37and SEQ ID NO:105; (vii) SEQ ID NO:38 and SEQ ID NO:105; (viii) SEQ IDNO:39 and SEQ ID NO:104; (ix) SEQ ID NO:39 and SEQ ID NO:105; (x) SEQ IDNO:39 and SEQ ID NO:106; (xi) SEQ ID NO:41 and SEQ ID NO:104; (xii) SEQID NO:41 and SEQ ID NO:106; (xiii) SEQ ID NO:43 and SEQ ID NO:106; (xiv)SEQ ID NO:45 and SEQ ID NO:103; (xv) SEQ ID NO:45 and SEQ ID NO:105;(xvi) SEQ ID NO:45 and SEQ ID NO:106; (xvii) SEQ ID NO:46 and SEQ IDNO:106; (xviii) SEQ ID NO:47 and SEQ ID NO:104; and (xix) SEQ ID NO:47and SEQ ID NO:106. In some aspects, the detecting step comprisescontacting the in vitro amplification reaction with at least onedetection probe oligomer, wherein the detection probe oligomercomprises: (i) a target hybridizing sequence and, optionally, one ormore nucleobases that are not complementary to the Zika virus targetnucleic acid, and (ii) a detectable label. In some aspects, the targethybridizing sequence of the detection probe oligomer is from 17 to 23contiguous nucleobases in length. In some aspects, the targethybridizing sequence of the detection probe oligomer is selected fromthe group consisting of: SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NOs:66to 73. In some aspects, the target hybridizing sequence of the detectionprobe oligomer comprises the one or more nucleobases that are notcomplementary to the target nucleic acid. In some aspects, the targethybridizing sequence of the detection probe oligomer comprises at eitherits 3′ end or its 5′ end from 3 to 7 contiguous nucleobases that are notcomplementary to the target sequence of the Zika virus nucleic acid. Insome aspects, the 3 to 7 contiguous nucleobases that are notcomplementary to the target sequence, are complementary to a portion ofthe target hybridizing sequence of the detection probe oligomer. In someaspects, the detectable label is a fluorescent label, a luminescentlabel, a chromophore label, a radionuclide label, a ligand label, anenzyme label, or a reactive group label. In some aspects, the detectablelabel is a chemiluminescent label. In some aspects, the detectable labelis an acridinium ester. In some aspects, the detectable label is afluorescent label and wherein the detection probe oligomer furthercomprises a quenching compound. In some aspects, the detection probeoligomer is selected from the group consisting of TaqMan detectionprobes, molecular beacons, and molecular torches. In some aspects, thetarget hybridizing sequence does not comprise the one or morenucleobases that are not complementary to the target nucleic acid. Insome aspects, the combination of the first amplification oligomer andthe second amplification oligomer is selected from the group consistingof: (i) SEQ ID NO:36 and SEQ ID NO:103; (ii) SEQ ID NO:36 and SEQ IDNO:104; (iii) SEQ ID NO:36 and SEQ ID NO:105; (iv) SEQ ID NO:37 and SEQID NO:102; (v) SEQ ID NO:37 and SEQ ID NO:103; (vi) SEQ ID NO:37 and SEQID NO:105; (vii) SEQ ID NO:38 and SEQ ID NO:105; (viii) SEQ ID NO:39 andSEQ ID NO:104; (ix) SEQ ID NO:39 and SEQ ID NO:105; (x) SEQ ID NO:39 andSEQ ID NO:106; (xi) SEQ ID NO:41 and SEQ ID NO:104; (xii) SEQ ID NO:41and SEQ ID NO:106; (xiii) SEQ ID NO:43 and SEQ ID NO:106; (xiv) SEQ IDNO:45 and SEQ ID NO:103; (xv) SEQ ID NO:45 and SEQ ID NO:105; (xvi) SEQID NO:45 and SEQ ID NO:106; (xvii) SEQ ID NO:46 and SEQ ID NO:106;(xviii) SEQ ID NO:47 and SEQ ID NO:104; and (xix) SEQ ID NO:47 and SEQID NO:106, and wherein the detection probe oligomer comprises a targethybridizing sequence consisting of SEQ ID NO:61, and the detectablelabel consists of an acridinium ester label. In some aspects, theamplification step (b) is performed with the detection step (c). In someaspects, before the amplification step (b), the method further comprisespurifying the Zika virus target sequence away from one or morecomponents of the sample. In some aspects, the purifying step comprisescontacting the sample with at least one capture probe oligomercomprising a target-hybridizing sequence covalently attached to a moietythat binds to an immobilized probe, wherein the target-hybridizingsequence is selected from the group consisting of SEQ ID NOs:166 to 178.In some aspects, the moiety comprises a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO:181 and SEQ ID NO:182. In someaspects, the immobilized probe comprises a nucleic acid sequenceconsisting of SEQ ID NO:180. In some aspects, the capture probe oligomercomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NOs:153 to 165. In some aspects, the in vitro amplificationreaction is an isothermal amplification reaction. In some aspects, thein vitro amplification reaction is a TMA reaction.

Further disclosed herein are multiplex methods for determining thepresence or absence of a Zika virus nucleic acid in a sample, the methodcomprising the steps of: (A) contacting a sample with at least twocombinations of at least two amplification oligomers, each foramplifying a separate target sequence of a Zika virus target nucleicacid, wherein comprising at least two combinations of at least twoamplification oligomers selected from the group consisting of: (1) (i)at least two amplification oligomers for amplifying a target sequence ofa Zika virus target nucleic acid, wherein the target sequence consistsessentially of SEQ ID NO:189, accounting for complements thereof, RNAequivalents thereof, or both, wherein the first amplification oligomerand the second amplification oligomer are configured to hybridize toopposite ends of the target sequence, to generate amplificationproducts; wherein a first amplification oligomer comprises a targethybridizing sequence that is from 17 to 26 contiguous nucleobases inlength, and wherein a second amplification oligomer comprises a targethybridizing sequence that is from 17 to 26 contiguous nucleobases inlength and, optionally, joined at its 5′ end to a promoter sequence,combined with (ii) at least two amplification oligomers for amplifying aZika virus nucleic acid in a sample, the oligomer combinationcomprising: at least two amplification oligomers for amplifying a targetsequence of a Zika virus target nucleic acid, wherein the targetsequence consists essentially of SEQ ID NO:187, accounting forcomplements thereof, RNA equivalents thereof, or both, wherein the firstamplification oligomer and the second amplification oligomer areconfigured to hybridize to opposite ends of the target sequence,including complements thereof and/or RNA equivalents thereof, togenerate amplification products; wherein a first amplification oligomercomprises a target hybridizing sequence that is from 19 to 23 contiguousnucleobases in length, and wherein a second amplification oligomercomprises a target hybridizing sequence that is from 19 to 25 contiguousnucleobases in length and, optionally, joined at its 5′ end to apromoter sequence; (2) (i) at least two amplification oligomers foramplifying a target sequence of a Zika virus target nucleic acid,wherein the target sequence consists essentially of SEQ ID NO:189,accounting for complements thereof, RNA equivalents thereof, or both,wherein the first amplification oligomer and the second amplificationoligomer are configured to hybridize to opposite ends of the targetsequence, to generate amplification products; wherein a firstamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 26 contiguous nucleobases in length, and wherein a secondamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 26 contiguous nucleobases in length and, optionally, joinedat its 5′ end to a promoter sequence, combined with (ii) at least twoamplification oligomers for amplifying a Zika virus nucleic acid in asample, the oligomer combination comprising: at least two amplificationoligomers for amplifying a target sequence of a Zika virus targetnucleic acid, wherein the target sequence consists essentially of SEQ IDNO:188, accounting for complements thereof, RNA equivalents thereof, orboth, wherein the first amplification oligomer and the secondamplification oligomer are configured to hybridize to opposite ends ofthe target sequence, including complements thereof and/or RNAequivalents thereof, to generate amplification products; wherein a firstamplification oligomer comprises a target hybridizing sequence that isfrom 19 to 23 contiguous nucleobases in length, and wherein a secondamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 23 contiguous nucleobases in length and, optionally, joinedat its 5′ end to a promoter sequence; (3) (i) at least two amplificationoligomers for amplifying a Zika virus nucleic acid in a sample, theoligomer combination comprising: at least two amplification oligomersfor amplifying a target sequence of a Zika virus target nucleic acid,wherein the target sequence consists essentially of SEQ ID NO:187,accounting for complements thereof, RNA equivalents thereof, or both,wherein the first amplification oligomer and the second amplificationoligomer are configured to hybridize to opposite ends of the targetsequence, including complements thereof and/or RNA equivalents thereof,to generate amplification products; wherein a first amplificationoligomer comprises a target hybridizing sequence that is from 19 to 23contiguous nucleobases in length, and wherein a second amplificationoligomer comprises a target hybridizing sequence that is from 19 to 25contiguous nucleobases in length and, optionally, joined at its 5′ endto a promoter sequence, combined with (ii) at least two amplificationoligomers for amplifying a Zika virus nucleic acid in a sample, theoligomer combination comprising: at least two amplification oligomersfor amplifying a target sequence of a Zika virus target nucleic acid,wherein the target sequence consists essentially of SEQ ID NO:188,accounting for complements thereof, RNA equivalents thereof, or both,wherein the first amplification oligomer and the second amplificationoligomer are configured to hybridize to opposite ends of the targetsequence, including complements thereof and/or RNA equivalents thereof,to generate amplification products; wherein a first amplificationoligomer comprises a target hybridizing sequence that is from 19 to 23contiguous nucleobases in length, and wherein a second amplificationoligomer comprises a target hybridizing sequence that is from 17 to 23contiguous nucleobases in length and, optionally, joined at its 5′ endto a promoter sequence; and (4) (i) at least two amplification oligomersfor amplifying a target sequence of a Zika virus target nucleic acid,wherein the target sequence consists essentially of SEQ ID NO:189,accounting for complements thereof, RNA equivalents thereof, or both,wherein the first amplification oligomer and the second amplificationoligomer are configured to hybridize to opposite ends of the targetsequence, to generate amplification products; wherein a firstamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 26 contiguous nucleobases in length, and wherein a secondamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 26 contiguous nucleobases in length and, optionally, joinedat its 5′ end to a promoter sequence, combined with (ii) at least twoamplification oligomers for amplifying a Zika virus nucleic acid in asample, the oligomer combination comprising: at least two amplificationoligomers for amplifying a target sequence of a Zika virus targetnucleic acid, wherein the target sequence consists essentially of SEQ IDNO:187, accounting for complements thereof, RNA equivalents thereof, orboth, wherein the first amplification oligomer and the secondamplification oligomer are configured to hybridize to opposite ends ofthe target sequence, including complements thereof and/or RNAequivalents thereof, to generate amplification products; wherein a firstamplification oligomer comprises a target hybridizing sequence that isfrom 19 to 23 contiguous nucleobases in length, and wherein a secondamplification oligomer comprises a target hybridizing sequence that isfrom 19 to 25 contiguous nucleobases in length and, optionally, joinedat its 5′ end to a promoter sequence, and combined with (iii) at leasttwo amplification oligomers for amplifying a Zika virus nucleic acid ina sample, the oligomer combination comprising: at least twoamplification oligomers for amplifying a target sequence of a Zika virustarget nucleic acid, wherein the target sequence consists essentially ofSEQ ID NO:188, accounting for complements thereof, RNA equivalentsthereof, or both, wherein the first amplification oligomer and thesecond amplification oligomer are configured to hybridize to oppositeends of the target sequence, including complements thereof and/or RNAequivalents thereof, to generate amplification products; wherein a firstamplification oligomer comprises a target hybridizing sequence that isfrom 19 to 23 contiguous nucleobases in length, and wherein a secondamplification oligomer comprises a target hybridizing sequence that isfrom 17 to 23 contiguous nucleobases in length and, optionally, joinedat its 5′ end to a promoter sequence; (B) performing an in vitro nucleicacid amplification reaction, wherein for (A)(1) the at least twoamplification oligomer of (i) and/or (ii) hybridize their respectivetarget sequence present in the sample to generate an amplificationproduct(s); or wherein for (A)(2) the at least two amplificationoligomer of (i) and/or (ii) hybridize their respective target sequencepresent in the sample to generate an amplification product(s); orwherein for (A)(3) the at least two amplification oligomer of (i) and/or(ii) hybridize their respective target sequence present in the sample togenerate an amplification product(s); or wherein for (A)(4) the at leasttwo amplification oligomer of (i) and/or (ii) and/or (iii) hybridizetheir respective target sequence present in the sample to generate anamplification product(s); and (C) detecting the presence or absence ofthe amplification product(s), thereby determining the presence orabsence of the Zika virus nucleic acid in the sample. In some aspects,the detecting step comprises contacting the in vitro amplificationreaction with at least one detection probe oligomer, wherein thedetection probe oligomer comprises: (i) a target hybridizing sequenceconfigured to hybridize under stringent conditions to a target sequenceof a Zika virus nucleic acid, and, optionally, one or more nucleobasesthat are not complementary to the Zika virus target nucleic acid, and(ii) a detectable label, wherein the Zika virus target nucleic acidsequence is selected from the group consisting of: SEQ ID NO:187, SEQ IDNO:188, & SEQ ID NO:189, including a complement thereof, and/or an RNAequivalent thereof. In some aspects, the detection probe oligomercomprises a target hybridizing sequence that is configured toselectively hybridize a sequence selected from the group consisting of:SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, & SEQ IDNO:198, including a complement thereof, and/or an RNA equivalentthereof. In some aspects, the target hybridizing sequence of thedetection probe oligomer is from 17 to 23 contiguous nucleobases inlength. In some aspects, the target hybridizing sequence of thedetection probe oligomer is selected from the group consisting of: SEQID NOs:54 to 63 and 66 to 73. In some aspects the combinations ofamplification oligomers and detection probe oligomers are: for(A)(1)(i), (A)(2)(i) or (A)(4)(i) the detection probe oligomer comprisesa nucleic acid sequence selected from the group consisting of SEQ IDNOs:56, 57, & 64 to 73; for (A)(1)(ii), (A)(3)(i), or (A)(4)(ii) thedetection probe oligomer comprises a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs:54, 55, & 58; or for (A)(2)(ii)m(A)(3)(ii), or (A)(4)(iii) the detection probe oligomer is selected fromthe group consisting of SEQ ID NOs:59 to 63. In some aspects, thedetection probe oligomer is selected from the group consisting of TaqMandetection probes, molecular beacons, and molecular torches. In someaspects, the detectable label is a fluorescent label, a luminescentlabel, a chromophore label, a radionuclide label, a ligand label, anenzyme label, or a reactive group label. In some aspects, the detectablelabel is a chemiluminescent label. In some aspects, the detectable labelis an acridinium ester. In some aspects, the detectable label is afluorescent label and wherein the detection probe oligomer furthercomprises a quenching compound. In some aspects, the amplification step(b) is performed with the detection step (c). In some aspects, beforethe amplification step (b), the method further comprises purifying theZika virus target sequence away from one or more components of thesample. In some aspects, the purifying step comprises contacting thesample with at least one capture probe oligomer comprising atarget-hybridizing sequence covalently attached to a moiety that bindsto an immobilized probe, wherein the target-hybridizing sequence isselected from the group consisting of SEQ ID NOs:166 to 178. In someaspects, the moiety comprises a nucleic acid sequence selected from thegroup consisting of SEQ ID NO:181 and SEQ ID NO:182; wherein theimmobilized probe comprises a nucleic acid sequence consisting of SEQ IDNO:180; or both. In some aspects, the capture probe oligomer comprises anucleic acid sequence selected from the group consisting of SEQ IDNOs:153 to 165. In some aspects, the in vitro amplification reaction isan isothermal amplification reaction. In some aspects, the in vitroamplification reaction is a TMA reaction. These and other aspects willbecome evident upon reference to the following detailed description andthe attached drawings.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art pertinent to the methods and compositions described. As usedherein, the following terms and phrases have the meanings ascribed tothem unless specified otherwise.

The terms “a,” “an,” and “the” include plural referents, unless thecontext clearly indicates otherwise. For example, “a nucleic acid” asused herein is understood to represent one or more nucleic acids. Assuch, the terms “a” (or “an”), “one or more,” and “at least one” can beused interchangeably herein.

“Sample” includes any specimen that may contain Zika virus or componentsthereof, such as nucleic acids or fragments of nucleic acids. For theavoidance of doubt, a medium containing a synthetic in vitro transcriptcomprising a Zika virus nucleic acid is considered a sample. Samplesinclude “biological samples” which include any material derived from aliving or dead human that may contain Zika virus or target nucleic acidderived therefrom. Biological samples include, but are not limited to,whole blood, red blood cells, peripheral blood, plasma, serum, lymphnode, gastrointestinal tissue (e.g., liver), material collected using avaginal swab, material collected using a cervical brush, materialcollected via bronchoscopy, material collected via bronchoaveolarlavage, tissue, sputum, saliva or other body fluids or materials. Also,samples may include processed or purified samples, such as thoseobtained from passing samples over or through a filtering device, orcapturing a nucleic acid of interest in a target capture reaction, orfollowing centrifugation, or by adherence to a medium, matrix, orsupport.

“Nucleic acid” refers to a multimeric compound comprising two or morecovalently bonded nucleosides and/or nucleoside analogs and/or baseanalogs, linked together by phosphodiester bonds or other linkages toform a polynucleotide. Sugar moieties of the nucleic acid may be riboseand/or deoxyribose. Sugar moieties may comprise substitutions such as,for example, 2′-methoxy substitutions and 2′-halide substitutions (e.g.,2′-F). Synthetic methods for making nucleic acids in vitro arewell-known in the art although nucleic acids may be purified fromnatural sources using routine techniques.

The term “polynucleotide” as used herein denotes a nucleic acid chain.Throughout this application, nucleic acids are designated by the5′-terminus to the 3′-terminus. Synthetic nucleic acids, e.g., DNA, RNA,DNA/RNA chimerics, (including when non-natural nucleotides or analoguesare included therein), are typically synthesized “3′-to-5′,” i.e., bythe addition of nucleotides to the 5′-terminus of a growing nucleicacid.

A “target nucleic acid” as used herein is a nucleic acid comprising atarget sequence to be amplified. Target nucleic acids may be DNA or RNAas described herein, and may be either single-stranded ordouble-stranded. Target nucleic acids may be any of the nucleic acidobtained directly from a sample, a synthetic nucleic acid construct, anda nucleic acid amplification product. The target nucleic acid mayinclude other sequences besides the target sequence, which may not beamplified. For example, a target nucleic acid can be an entire genome, agene, a region within a genome, an expression product, a chimericnucleic acid, an amplification product, or a target sequence.

The term “target sequence” as used herein refers to the particularnucleotide sequence of the target nucleic acid that is to be amplifiedand/or detected. The “target sequence” includes the complexing sequencesto which oligonucleotides (e.g., priming oligonucleotides and/orpromoter oligonucleotides) complex during an amplification processes.Where the target nucleic acid is originally single-stranded, the term“target sequence” will also refer to the sequence complementary to the“target sequence” as present in the target nucleic acid. Where thetarget nucleic acid is originally double-stranded, the term “targetsequence” refers to both the sense (+) and antisense (−) strands. Atarget sequence consisting essentially of a certain SEQ ID NO (e.g., SEQID NOs:187, 188 or 189) means that the target sequence may have from 0to 20 fewer nucleotides on one or both ends of the referenced sequence.Preferably, a target sequence consisting essentially of SEQ ID NO:189has from 0 to 3 fewer nucleotides on the 5′ end and from 0 to 8 fewernucleotides on the 3′ end compared SEQ ID NO:189. Preferably, a targetsequence consisting essentially of SEQ ID NO:187 has from 0 to 6 fewernucleotides on the 5′ end compared to SEQ ID NO:187. Preferably, atarget sequence consisting essentially of SEQ ID NO:188 has from 0 to 4fewer nucleotides on the 3′ end compared to SEQ ID NO:188.

“Target-hybridizing sequence” is used herein to refer to the portion ofan oligomer that is configured to hybridize with a target nucleic acidsequence. Preferably, the target-hybridizing sequences are configured tospecifically hybridize with a target nucleic acid sequence.Target-hybridizing sequences may be 100% complementary to the portion ofthe target sequence to which they are configured to hybridize, but notnecessarily. Target-hybridizing sequences may also include inserted,deleted and/or substituted nucleotide residues relative to a targetsequence. Less than 100% complementarity of a target-hybridizingsequence to a target sequence may arise, for example, when the targetnucleic acid is a plurality strains within a species, such as would bethe case for an oligomer configured to hybridize to various genotypes ofZika virus species isolates. It is understood that other reasons existfor configuring a target-hybridizing sequence to have less than 100%complementarity to a target nucleic acid.

The term “targets a sequence” as used herein in reference to a region ofZika virus nucleic acid refers to a process whereby an oligonucleotidehybridizes to the target sequence in a manner that allows for targetcapture, amplification and/or detection as described herein. In onepreferred embodiment, the oligonucleotide is complementary with thetargeted Zika virus nucleic acid sequence and contains no mismatches. Inanother preferred embodiment, the oligonucleotide is complementary butcontains 1, 2, 3, 4, or 5 mismatches with the targeted Zika virusnucleic acid sequence. Preferably, the oligonucleotide that hybridizesto the Zika virus nucleic acid sequence includes at least 10 to as manyas 50 nucleotides complementary to the target sequence. It is understoodthat at least 10 and as many as 50 is an inclusive range such that 10,50 and each whole number there between are included. Preferably, theoligomer specifically hybridizes to the target sequence.

The term “configured to” denotes an actual arrangement of thepolynucleotide sequence configuration of a referenced oligonucleotidetarget-hybridizing sequence. For example, amplification oligomers thatare configured to generate a specified amplicon from a target sequencehave polynucleotide sequences that hybridize to the target sequence andcan be used in an amplification reaction to generate the amplicon. Alsoas an example, oligonucleotides that are configured to specificallyhybridize to a target sequence have a polynucleotide sequence thatspecifically hybridizes to the referenced sequence under stringenthybridization conditions.

The term “configured to specifically hybridize to” as used herein meansthat the target-hybridizing sequence of an amplificationoligonucleotide, detection probe, or other oligonucleotide is designedto have a polynucleotide sequence that could target a sequence of thereferenced Zika virus target nucleic acid. Such an oligonucleotide isnot limited to targeting that sequence only, but is rather useful as acomposition, in a kit, or in a method for targeting a Zika virus targetnucleic acid. The oligonucleotide is designed to function as a componentof an assay for amplification and detection of Zika virus from a sample,and therefore is designed to target Zika virus in the presence of othernucleic acids commonly found in testing samples. “Specifically hybridizeto” does not mean exclusively hybridize to, as some small level ofhybridization to non-target nucleic acids may occur, as is understood inthe art. Rather, “specifically hybridize to” means that theoligonucleotide is configured to function in an assay to primarilyhybridize the target so that an accurate detection of target nucleicacid in a sample can be determined.

The interchangeable terms “oligomer,” “oligo,” and “oligonucleotide”refer to a nucleic acid having generally less than 1,000 nucleotide (nt)residues, including polymers in a range having a lower limit of about 5nt residues and an upper limit of about 900 nt residues, including allwhole numbers therein. Oligonucleotides may be purified from naturallyoccurring sources or may be synthesized using any of a variety ofwell-known enzymatic or chemical methods. The term oligonucleotide doesnot denote any particular function to the reagent; rather, it is usedgenerically to cover all such reagents described herein. Anoligonucleotide may serve various different functions. For example, itmay function as a primer if it is specific for and capable ofhybridizing to a complementary strand and can further be extended in thepresence of a nucleic acid polymerase; it may function as a primer andprovide a promoter if it contains a sequence recognized by an RNApolymerase and allows for transcription (e.g., a T7 Primer); and it mayfunction to detect a target nucleic acid if it is capable of hybridizingto the target nucleic acid, or an amplicon thereof, and further providesa detectable moiety (e.g., an acridinium-ester compound).

As used herein, an oligonucleotide “substantially corresponding to” aspecified reference nucleic acid sequence means that the oligonucleotideis sufficiently similar to the reference nucleic acid sequence such thatthe oligonucleotide has similar hybridization properties to thereference nucleic acid sequence in that it would hybridize with the sametarget nucleic acid sequence under stringent hybridization conditions.One skilled in the art will understand that “substantially correspondingoligonucleotides” can vary from a reference sequence and still hybridizeto the same reference sequence (e.g., a primer sequence hybridizing to atarget nucleic acid sequence). It is also understood that a firstnucleic acid corresponding to a second nucleic acid includes the RNA orDNA equivalent thereof as well as DNA/RNA chimerics thereof, andincludes the complements thereof, unless the context clearly dictatesotherwise. This variation from the nucleic acid may be stated in termsof a percentage of identical bases within the sequence or the percentageof perfectly complementary bases between the probe or primer and itstarget sequence. Thus, in certain embodiments, an oligonucleotide“substantially corresponds” to a reference nucleic acid sequence ifthese percentages of base identity or complementarity are from 100% toabout 80%. In preferred embodiments, the percentage is from 100% toabout 85%, including all whole and partial numbers therein. Similarly, aregion of a nucleic acid or amplified nucleic acid can be referred toherein as corresponding to a reference nucleic acid sequence. Oneskilled in the art will understand the various modifications to thehybridization conditions that might be required at various percentagesof complementarity to allow hybridization to a specific target sequencewithout causing an unacceptable level of non-specific hybridization.

As used herein, the phrase “or its complement, or an RNA equivalent, orDNA/RNA chimeric thereof,” with reference to a DNA sequence, includes(in addition to the referenced DNA sequence) the complement of the DNAsequence, an RNA equivalent of the referenced DNA sequence, an RNAequivalent of the complement of the referenced DNA sequence, a DNA/RNAchimeric of the referenced DNA sequence, and a DNA/RNA chimeric of thecomplement of the referenced DNA sequence. Similarly, the phrase “or itscomplement, or a DNA equivalent or DNA/RNA chimeric thereof,” withreference to an RNA sequence, includes (in addition to the referencedRNA sequence) the complement of the RNA sequence, a DNA equivalent ofthe referenced RNA sequence, a DNA equivalent of the complement of thereferenced RNA sequence, a DNA/RNA chimeric of the referenced RNAsequence, and a DNA/RNA chimeric of the complement of the referenced RNAsequence.

As used herein, a “blocking moiety” is a substance used to “block” the3′-terminus of an oligonucleotide or other nucleic acid so that itcannot be efficiently extended by a nucleic acid polymerase. Oligomersnot intended for extension by a nucleic acid polymerase may include ablocker group that replaces the 3′ OH to prevent enzyme-mediatedextension of the oligomer in an amplification reaction. For example,blocked amplification oligomers and/or detection probes present duringamplification may not have functional 3′ OH and instead include one ormore blocking groups located at or near the 3′ end. In some embodimentsa blocking group near the 3′ end and may be within five residues of the3′ end and is sufficiently large to limit binding of a polymerase to theoligomer. In other embodiments a blocking group is covalently attachedto the 3′ terminus. Many different chemical groups may be used to blockthe 3′ end, e.g., alkyl groups, non-nucleotide linkers, alkane-dioldideoxynucleotide residues, and cordycepin.

An “amplification oligomer” is an oligomer, at least the 3′-end of whichis complementary to a target nucleic acid, and which hybridizes to atarget nucleic acid, or its complement, and participates in a nucleicacid amplification reaction. The nucleic acid amplification reaction canbe through an extension of the 3′ end of the amplification oligomer. Theamplification can also be through a promoter sequence attached to theamplification oligomer. Examples of amplification oligomers include, butare not limited to, primers and promoter-based amplification oligomers.Promoter-based amplification oligomers are referred to as promoterprimers, promoter providers, T7 amplification oligomers, T7 providersand T7 primers.

As used herein, a “primer” is an amplification oligomer that hybridizesto a target nucleic acid and contains a 3′ OH end that is extended by apolymerase in an amplification process.

As used herein, a “promoter-based amplification oligomer,” referred toalso as “promoter primers,” “promoter providers,” “T7 amplificationoligomers,” “T7 providers” and “T7 primers,” refers to anoligonucleotide comprising at least first and second regions. The “firstregion” of a promoter-based amplification oligonucleotide comprises abase sequence that hybridizes to a target sequence (e.g., atarget-hybridizing sequence), where the first region is situated 3′, butnot necessarily adjacent to, a second region that is a promotersequence. In some instances, the 3′ end of the first region is notextended by a polymerase, and the promoter-based amplification oligomerparticipates in or facilitates amplification by the promoter sequence.In certain instances, there is an intervening sequence or sequencesbetween the target-hybridizing sequence at the 3′ end of theamplification oligomer and the promoter sequence at the 5′ end of theamplification oligomer. One example of an intervening sequence is a tagsequence that is useful as an added sequence in an amplificationproduct.

As used herein, a “promoter” is a specific nucleic acid sequence that isrecognized by a DNA-dependent RNA polymerase (“transcriptase”) as asignal to bind to the nucleic acid and begin the transcription of RNA ata specific site.

“Amplification” refers to any known procedure for obtaining multiplecopies of a target nucleic acid sequence or its complement or fragmentsthereof. The multiple copies may be referred to as amplicons oramplification products. Known amplification methods include, forexample, replicase-mediated amplification (see, e.g., U.S. Pat. No.4,786,600), polymerase chain reaction (PCR; see, e.g., U.S. Pat. Nos.4,683,195; 4,683,202; and 4,800,159), ligase chain reaction (LCR; see,e.g., U.S. Pat. Nos. 5,427,930 and 5,516,663), strand-displacementamplification (SDA; see, e.g., U.S. Pat. Nos. 5,422,252; 5,547,861; and5,648,211), and transcription-mediated or transcription-associatedamplification.

“Transcription-associated amplification” or “transcription-mediatedamplification” (TMA) refer to nucleic acid amplification that uses anRNA polymerase to produce multiple RNA transcripts from a nucleic acidtemplate. These methods generally employ an RNA polymerase, a DNApolymerase, deoxyribonucleoside triphosphates, ribonucleosidetriphosphates, and a template complementary oligonucleotide thatincludes a promoter sequence, and optionally may include one or moreother oligonucleotides. TMA methods and single-primertranscription-associated amplification methods are embodiments ofamplification methods used for detection of Zika virus target sequencesas described herein. Variations of transcription-associatedamplification are well-known in the art as previously disclosed indetail (see, e.g., U.S. Pat. Nos. 4,868,105; 5,124,246; 5,130,238;5,399,491; 5,437,990; 5,554,516; and 7,374,885; and International PatentApplication Pub. Nos. WO 88/01302; WO 88/10315; and WO 95/03430). Theperson of ordinary skill in the art will appreciate that the disclosedcompositions may be used in amplification methods based on extension ofoligomer sequences by a polymerase.

As used herein, the term “real-time TMA” refers to single-primertranscription-mediated amplification (“TMA”) of target nucleic acid thatis monitored by real-time detection means.

The term “amplicon” or “amplification product” as used herein refers tothe nucleic acid molecule generated during an amplification procedurethat is complementary or homologous to a sequence contained within thetarget sequence. The complementary or homologous sequence of an ampliconis sometimes referred to herein as a “target-specific sequence.”Amplicons generated using the amplification oligomers described hereinmay comprise non-target specific sequences, such as those added by a tagsequence on an amplification oligomer, and/or modified nucleotidesincluded in the amplification oligomers or in the dNTP mixture.Amplicons can be double-stranded or single-stranded and can include DNA,RNA, or both. For example, DNA-dependent RNA polymerase transcribessingle-stranded amplicons from double-stranded DNA duringtranscription-mediated amplification procedures. These single-strandedamplicons are RNA amplicons and can be either strand of adouble-stranded complex, depending on how the amplification oligomersare configured. Thus, amplicons can be single-stranded RNA.RNA-dependent DNA polymerases synthesize a DNA strand that iscomplementary to an RNA template. Thus, amplicons can be double-strandedDNA and RNA hybrids. RNA-dependent DNA polymerases often include RNaseactivity, or are used in conjunction with an RNase, which degrades theRNA strand. Thus, amplicons can be single stranded DNA. RNA-dependentDNA polymerases and DNA-dependent DNA polymerases synthesizecomplementary DNA strands from DNA templates. Thus, amplicons can bedouble-stranded DNA. RNA-dependent RNA polymerases synthesize RNA froman RNA template. Thus, amplicons can be double-stranded RNA.DNA-dependent RNA polymerases synthesize RNA from double-stranded DNAtemplates, also referred to as transcription. Thus, amplicons can besingle stranded RNA. Amplicons and methods for generating amplicons areknown to those skilled in the art. For convenience herein, a singlestrand of RNA or a single strand of DNA may represent an amplicongenerated by an amplification oligomer combination described herein.Such representation is not meant to limit the amplicon to therepresentation shown. Skilled artisans in possession of the instantdisclosure will use amplification oligomers and polymerase enzymes togenerate any of the numerous types of amplicons, all within the spiritand scope of the current disclosure.

A “non-target-specific sequence,” as is used herein refers to a regionof an oligomer sequence, wherein said region does not stably hybridizewith a target sequence under standard hybridization conditions.Oligomers with non-target-specific sequences include, but are notlimited to, promoter primers (e.g., the 5′ promoter sequence of theamplification oligomer) and molecular beacons and torches (e.g., one orboth of the stem members).

“Detection probe,” “detection oligonucleotide,” and “detection probeoligomer” are used interchangeably to refer to a nucleic acid oligomerthat hybridizes specifically to a target sequence in a nucleic acid, orin an amplified nucleic acid, under conditions that promotehybridization to allow detection of the target sequence or amplifiednucleic acid. Detection may either be direct (e.g., a probe hybridizeddirectly to its target sequence) or indirect (e.g., a probe linked toits target via an intermediate molecular structure). Detection probesmay be DNA, RNA, analogs thereof or combinations thereof (e.g., DNA/RNAchimerics) and they may be labeled or unlabeled. Detection probes mayfurther include modifications such as, e.g., 2′-O-methyl linkages. Adetection probe may comprise target-specific sequences and othersequences that contribute to the three-dimensional conformation of theprobe (see, e.g., U.S. Pat. Nos. 5,118,801; 5,312,728; 6,849,412;6,835,542; 6,534,274; and 6,361,945; and US Patent Application Pub. No.20060068417).

As used herein, a “label” refers to a moiety or compound joined directlyor indirectly to a probe that is detected or leads to a detectablesignal. Direct labeling can occur through bonds or interactions thatlink the label to the probe. Indirect labeling can occur through use ofa bridging moiety or “linker” such as a binding pair member, an antibodyor additional oligomer, which is either directly or indirectly labeled,and which may amplify the detectable signal. Labels include anydetectable moiety, such as a radionuclide, ligand (e.g., biotin,avidin), enzyme or enzyme substrate, reactive group, or chromophore(e.g., dye, particle, or bead that imparts detectable color),luminescent compound (e.g., bioluminescent, phosphorescent, orchemiluminescent labels), or fluorophore. Labels may be detectable in ahomogeneous assay see, e.g., U.S. Pat. Nos. 5,283,174; 5,656,207; and5,658,737. Labels include chemiluminescent compounds, e.g., acridiniumester (“AE”) compounds that include standard AE and derivatives (see,e.g., U.S. Pat. Nos. 5,656,207; 5,658,737; and 5,639,604). Synthesis andmethods of attaching labels to nucleic acids and detecting labels arewell known. (See, e.g., Sambrook et al., Molecular Cloning, A LaboratoryManual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N Y, 1989, Chapter 10, incorporated by reference herein. Seealso U.S. Pat. Nos. 5,658,737; 5,656,207; 5,547,842; 5,283,174; and4,581,333). More than one label, and more than one type of label, may bepresent on a particular probe, or detection may use a mixture of probesin which each probe is labeled with a compound that produces adetectable signal (see, e.g., U.S. Pat. Nos. 6,180,340 and 6,350,579).

“Capture probe,” “capture oligonucleotide,” “target captureoligonucleotide,” and “capture probe oligomer” are used interchangeablyto refer to a nucleic acid oligomer that specifically hybridizes to atarget sequence in a target nucleic acid by standard base pairing andjoins to a binding partner on an immobilized probe to capture the targetnucleic acid to a support. One example of a capture oligomer includestwo binding regions: a target sequence-binding region (e.g.,target-hybridizing sequence) and an immobilized probe-binding region,usually on the same oligomer, although the two regions may be present ontwo different oligomers joined together by one or more linkers. Anotherembodiment of a capture oligomer uses a target-sequence binding regionthat includes random or non-random poly-GU, poly-GT, or poly U sequencesto bind non-specifically to a target nucleic acid and link it to animmobilized probe on a support.

As used herein, an “immobilized oligonucleotide,” “immobilized probe,”or “immobilized nucleic acid” refers to a nucleic acid binding partnerthat directly or indirectly joins a capture oligomer to a solid support.An immobilized probe joined to a solid support facilitates separation ofa capture probe bound target from unbound material in a sampleImmobilized probes, solid supports and capture oligomers are describedin the literature (e.g., U.S. Pat. No. 6,110,678).

By “complementary” is meant that the nucleotide sequences of similarregions of two single-stranded nucleic acids, or two different regionsof the same single-stranded nucleic acid, have a nucleotide basecomposition that allow the single-stranded regions to hybridize togetherin a stable double-stranded hydrogen-bonded region under stringenthybridization or amplification conditions. Sequences that hybridize toeach other may be completely complementary or partially complementary tothe intended target sequence by standard nucleic acid base pairing(e.g., G:C, A:T, or A:U pairing). By “sufficiently complementary” ismeant a contiguous sequence that is capable of hybridizing to anothersequence by hydrogen bonding between a series of complementary bases,which may be complementary at each position in the sequence by standardbase pairing or may contain one or more residues, including abasicresidues that are not complementary. Sufficiently complementarycontiguous sequences typically are at least 80% (including all whole andpartial numbers from 80% to just less than 100%) complementary to asequence to which an oligomer is intended to specifically hybridize andthat stably hybridize with its target sequence under appropriatehybridization conditions, (see, e.g., Sambrook et al., MolecularCloning, A Laboratory Manual, 2^(nd) ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989 at §§ 1.90-1.91, 7.37-7.57,9.47-9.51 and 11.47-11.57, particularly §§ 9.50-9.51, 11.12-11.13,11.45-11.47 and 11.55-11.57).

By “preferentially hybridize” or “specifically hybridize” is meant thatunder stringent hybridization assay conditions, probes hybridize totheir target sequences, or replicates thereof, to form stableprobe:target hybrids, while at the same time formation of stableprobe:non-target hybrids is minimized Thus, a probe hybridizes to atarget sequence or replicate thereof to a sufficiently greater extentthan to a non-target sequence, to enable one having ordinary skill inthe art to accurately detect or quantitate RNA replicates orcomplementary DNA (cDNA) of the target sequence formed during theamplification. Appropriate hybridization conditions are well-known inthe art, may be predicted based on sequence composition, or can bedetermined by using routine testing methods (see, e.g., Sambrook et al.,Molecular Cloning, A Laboratory Manual, 2^(nd) ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N Y, 1989 at §§ 1.90-1.91,7.37-7.57, 9.47-9.51 and 11.47-11.57, particularly §§ 9.50-9.51,11.12-11.13, 11.45-11.47 and 11.55-11.57, incorporated by referenceherein).

“Sample preparation” refers to any steps or method that treats a samplefor subsequent amplification and/or detection of Zika virus nucleicacids present in the sample. Sample preparation may includephysical/mechanical disruption and/or chemical lysis of cellularcomponents to release intracellular components into a substantiallyaqueous or organic phase and removal of debris, such as by usingfiltration, centrifugation or adsorption. Sample preparation may includeuse of a target capture oligonucleotide that selectively ornon-specifically capture a target nucleic acid and separate it fromother sample components (e.g., as described in U.S. Pat. No. 6,110,678and International Patent Application Pub. No. WO 2008/016988). Samplepreparation can include pooling a plurality of samples into a singlepooled batch. Preferably for pooling, an aliquot of each sample ispooled into the larger batch. More preferably for pooling, an aliquot ofeach sample is first lysed and then pooled into the larger batch. Thelarger batch of pooled samples can be from a plurality of sampleswherein the plurality is from 2 to about 200 individual samples.

“Separating” or “purifying” means that one or more components of asample are removed or separated from other sample components. Samplecomponents include target nucleic acids usually in a generally aqueoussolution phase, which may also include cellular fragments, proteins,carbohydrates, lipids, and other nucleic acids. “Separating” or“purifying” does not connote any degree of purification. Typically,separating or purifying removes at least 70%, or at least 80%, or atleast 95% of the target nucleic acid from other sample components.

As used herein, a “DNA-dependent DNA polymerase” is an enzyme thatsynthesizes a complementary DNA copy from a DNA template. All knownDNA-dependent DNA polymerases require a complementary primer to initiatesynthesis. It is known that under suitable conditions a DNA-dependentDNA polymerase may synthesize a complementary DNA copy from an RNAtemplate. RNA-dependent DNA polymerases typically also haveDNA-dependent DNA polymerase activity.

As used herein, a “DNA-dependent RNA polymerase” or “transcriptase” isan enzyme that synthesizes multiple RNA copies from a double-stranded orpartially double-stranded DNA molecule having a promoter sequence thatis usually double-stranded. The RNA molecules (“transcripts”) aresynthesized in the 5′-to-3′ direction beginning at a specific positionjust downstream of the promoter.

As used herein, an “RNA-dependent DNA polymerase” or “reversetranscriptase” (“RT”) is an enzyme that synthesizes a complementary DNAcopy from an RNA template. All known reverse transcriptases also havethe ability to make a complementary DNA copy from a DNA template; thus,they are both RNA- and DNA-dependent DNA polymerases. RTs may also havean RNAse H activity. A primer is required to initiate synthesis withboth RNA and DNA templates.

As used herein, a “selective RNAse” is an enzyme that degrades the RNAportion of an RNA:DNA duplex but not single-stranded RNA,double-stranded RNA or DNA. An exemplary selective RNAse is RNAse H.Enzymes possessing the same or similar activity as RNAse H may also beused. Selective RNAses may be endonucleases or exonucleases. Mostreverse transcriptase enzymes contain an RNAse H activity in addition totheir polymerase activities.

The term “specificity,” in the context of an amplification and/ordetection system, is used herein to refer to the characteristic of thesystem which describes its ability to distinguish between target andnon-target sequences dependent on sequence and assay conditions. Interms of nucleic acid amplification, specificity generally refers to theratio of the number of specific amplicons produced to the number ofside-products (e.g., the signal-to-noise ratio). In terms of detection,specificity generally refers to the ratio of signal produced from targetnucleic acids to signal produced from non-target nucleic acids.

The term “sensitivity” is used herein to refer to the precision withwhich a nucleic acid amplification reaction can be detected orquantitated. The sensitivity of an amplification reaction is generally ameasure of the smallest copy number of the target nucleic acid that canbe reliably detected in the amplification system, and will depend, forexample, on the detection assay being employed, and the specificity ofthe amplification reaction, e.g., the ratio of specific amplicons toside-products.

As used herein, the term “relative light unit” (“RLU”) is an arbitraryunit of measurement indicating the relative number of photons emitted bythe sample at a given wavelength or band of wavelengths. RLU varies withthe characteristics of the detection means used for the measurement.

“Sample Transport Solution” generally refers to a solution containing 15mM sodium phosphate monobasic, 15 mM sodium phosphate dibasic, 1 mMEDTA, 1 mM EGTA, and 110 mM lithium lauryl sulfate (LLS), at pH 6.7.

“Target Capture Reagent” generally refers to a solution containing 250mM HEPES, 310 mM lithium hydroxide, 1.88 M lithium chloride, 100 mMEDTA, at pH 6.4, and 250 μg/ml of magnetic particles (1 micronSERA-MAG{circumflex over ( )}TM MG-CM particles, GE HealthcareLifesciences) with dT₁₄ oligomers covalently bound thereto.

“Wash Solution” generally refers to a solution containing 10 mM HEPES,150 mM sodium chloride, 6.5 mM sodium hydroxide, 1 mM EDTA, 0.3% (v/v)ethanol, 0.02% (w/v) methyl paraben, 0.01% (w/v) propyl paraben, and0.1% (w/v) sodium lauryl sulfate, at pH 7.5.

“Probe Reagent” generally refers to a solution containing one or morelabeled detection probes in a solution made up of 100 mM lithiumsuccinate, 2% (w/v) LLS, 15 mM mercaptoethanesulfonate, 1.2 M lithiumchloride, 20 mM EDTA, and 3% (v/v) ethanol, at pH 4.7.

“Amplification Reagent” generally refers to a concentrated mixture mixedwith other reaction components to produce a mixture containing 47.6 mMNa-HEPES, 12.5 mM N-acetyl-L-cysteine, 2.5% TRITON™ X-100, 54.8 mM KCl,23 mM MgCl2, 3 mM NaOH, 0.35 mM of each dNTP (dATP, dCTP, dGTP, dTTP),7.06 mM rATP, 1.35 mM rCTP, 1.35 mM UTP, 8.85 mM rGTP, 0.26 mM Na2EDTA,5% v/v glycerol, 2.9% trehalose, 0.225% ethanol, 0.075% methylparaben,0.015% propylparaben, and 0.002% Phenol Red, at pH 7.5-7.6, althoughother formulations of amplification reagent may function equally well.Primers may be added to the amplification reagent or added toamplification reactions separate from the amplification reagent. Enzymesin an amplification reagent can include one or more of Moloney MurineLeukemia Virus Reverse Transcriptase (MMLV-RT) and bacteriophage T7 RNApolymerase for which units are functionally defined as: 1 U of MMLV-RTincorporates 1 nmol of dTTP in 10 min at 37° C. using 200-400 micromolaroligo dT-primed poly(A) as template, and 1 U of T7 RNA polymeraseincorporates 1 nmol of ATP into RNA in 1 hr at 37° C. using a DNAtemplate containing a T7 promoter.

“Hybridization Reagent” generally refers to a solution made up of 100 mMsuccinic acid, 2% (w/v) LLS, 100 mM lithium hydroxide, 15 mMaldrithiol-2, 1.2 M lithium chloride, 20 mM EDTA, and 3.0% (v/v)ethanol, at pH 4.7.

“Selection Reagent” generally refers to a solution containing 600 mMboric acid, 182.5 mM sodium hydroxide, 1% (v/v) octoxynol (TRITON®X-100), at pH 8.5.

“Detection Reagents” include “Detect Reagent I,” which generally refersto a solution containing 1 mM nitric acid and 32 mM hydrogen peroxide,and “Detect Reagent II,” which generally refers to a solution of 1.5 Msodium hydroxide.

DETAILED DESCRIPTION

Described herein are compositions, kits, and methods for amplifying anddetecting Zika virus nucleic acid from a sample. Preferably, the samplesare biological samples. The compositions, kits, and methods provideoligonucleotide sequences that recognize target sequences of the Zikavirus genome. Such oligonucleotides may be used as amplificationoligonucleotides. Other oligonucleotides may be used as probes fordetecting amplified sequences of Zika virus, or for capture of Zikavirus target nucleic acid.

The methods provide for the sensitive and specific detection of Zikavirus nucleic acids. The methods include performing a nucleic acidamplification of a Zika virus target nucleic acid and detecting theamplified product by, for example, specifically hybridizing theamplified product with a nucleic acid detection probe that provides asignal to indicate the presence of Zika virus in the sample. Theamplification step includes contacting the sample with one or moreamplification oligomers specific for a target sequence in a Zika virustarget nucleic acid to produce an amplified product if Zika virusnucleic acid is present in the sample. Amplification synthesizesadditional copies of the target sequence or its complement by using atleast one nucleic acid polymerase and an amplification oligomer toproduce the copies from a template strand (e.g., by extending thesequence from a primer using the template strand). One embodiment fordetecting the amplified product uses a hybridizing step that includescontacting the amplified product with at least one probe specific for asequence amplified by the selected amplification oligomers, e.g., asequence contained in the target sequence flanked by a pair of selectedamplification oligomers.

The detection step may be performed using any of a variety of knowntechniques to detect a signal specifically associated with the amplifiedtarget sequence, such as, e.g., by hybridizing the amplification productwith a labeled detection probe and detecting a signal resulting from thelabeled probe. The detection step may also provide additionalinformation on the amplified sequence, such as, e.g., all or a portionof its nucleic acid base sequence. Detection may be performed after theamplification reaction is completed, or may be performed simultaneouslywith amplifying the target sequence, e.g., in real time. In oneembodiment, the detection step allows homogeneous detection, e.g.,detection of the hybridized probe without removal of unhybridized probefrom the mixture (see, e.g., U.S. Pat. Nos. 5,639,604 and 5,283,174).

In embodiments that detect the amplified product near or at the end ofthe amplification step, a linear detection probe may be used to providea signal to indicate hybridization of the probe to the amplifiedproduct. One example of such detection uses a luminescentally labeledprobe that hybridizes to target nucleic acid. Luminescent label is thenhydrolyzed from non-hybridized probe. Detection is performed bychemiluminescence using a luminometer. (see, e.g., International PatentApplication Pub. No. WO 89/002476). In other embodiments that usereal-time detection, the detection probe may be a hairpin probe such as,for example, a molecular beacon, molecular torch, or hybridizationswitch probe that is labeled with a reporter moiety that is detectedwhen the probe binds to amplified product. Such probes may comprisetarget-hybridizing sequences and non-target-hybridizing sequences.Various forms of such probes have been described previously (see, e.g.,U.S. Pat. Nos. 5,118,801; 5,312,728; 5,925,517; 6,150,097; 6,849,412;6,835,542; 6,534,274; and 6,361,945; and US Patent Application Pub. Nos.20060068417A1 and 20060194240A1).

Preferred compositions described herein are configured to specificallyhybridize to Zika virus nucleic acids from one or more Zika virusstrains and, preferably with minimal cross-reactivity to other, non-Zikavirus nucleic acids suspected of being in a sample (e.g., other bloodborne pathogens). In some aspects, the disclosed compositions areconfigured to specifically hybridize to Zika virus nucleic acid withminimal cross-reactivity to one or more of hepatitis C virus (HCV),human immunodeficiency virus 1 (HIV 1), hepatitis B virus (HBV), Denguevirus plasmodium, babesia, and West Nile virus. In one aspect, thecompositions are part of a multiplex system that further includescomponents and methods for detecting one of more of these organisms.

SEQ ID NO: 1. (5′ to 3′) Gen Bank AccessionNumber/Version Number/GI Number AY632535.2 GI:226374362.agttgttgatctgtgtgagtcagactgcgacagttcgagtctgaagcgagagctaacaacagtatcaacaggtttaatttggatttggaaacgagagtttctggtcatgaaaaaccccaaagaagaaatccggaggatccggattgtcaatatgctaaaacgcggagtagcccgtgtaaaccccttgggaggtttgaagaggttgccagccggacttctgctgggtcatggacccatcagaatggattggcgatactagcctattgagatttacagcaatcaagccatcactgggccttatcaacagatggggttccgtggggaaaaaagaggctatggaaataataaagaagttcaagaaagatcttgctgccatgttgagaataatcaatgctaggaaagagaggaagagacgtggcgcagacaccagcatcggaatcattggcctcctgctgactacagccatggcagcagagatcactagacgcgggagtgcatactacatgtacttggataggagcgatgccgggaaggccatttcgtttgctaccacattgggagtgaacaagtgccacgtacagatcatggacctcgggcacatgtgtgacgccaccatgagttatgagtgccctatgctggatgagggagtggaaccagatgatgtcgattgctggtgcaacacgacatcaacttgggttgtgtacggaacctgtcatcacaaaaaaggtgaggcacggcgatctagaagagccgtgacgctcccttctcactctacaaggaagttgcaaacgcggtcgcagacctggttagaatcaagagaatacacgaagcacttgatcaaggttgaaaactggatattcaggaaccccgggtttgcgctagtggccgttgccattgcctggcttttgggaagctcgacgagccaaaaagtcatatacttggtcatgatactgctgattgccccggcatacagtatcaggtgcattggagtcagcaatagagacttcgtggagggcatgtcaggtgggacctgggttgatgagtcttggaacatggaggctgcgttaccgtgatggcacaggacaagccaacagtcgacatagagttggtcacgacgacggttagtaacatggccgaggtaagatcctattgctacgaggcatcgatatcggacatggcttcggacagtcgttgcccaacacaaggtgaagcctaccttgacaagcaatcagacactcaatatgtctgcaaaagaacattagtggacagaggttggggaaacggttgtggactttttggcaaagggagcttggtgacatgtgccaagtttacgtgttctaagaagatgaccgggaagagcattcaaccggaaaatctggagtatcggataatgctatcagtgcatggctcccagcatagcgggatgattggatatgaaactgacgaagatagagcgaaagtcgaggttacgcctaattcaccaagagcggaagcaaccttgggaggctttggaagcttaggacttgactgtgaaccaaggacaggccttgacttttcagatctgtattacctgaccatgaacaataagcattggttggtgcacaaagagtggtttcatgacatcccattgccttggcatgctggggcagacaccggaactccacactggaacaacaaagaggcattggtagaattcaaggatgcccacgccaagaggcaaaccgtcgtcgttctggggagccaggaaggagccgttcacacggctctcgctggagctctagaggctgagatggatggtgcaaagggaaggctgactctggccatttgaaatgccgcctaaaaatggacaagcttagattgaagggcgtgtcatattccttgtgcactgcggcattcacattcaccaaggtcccagctgaaacactgcatggaacagtcacagtggaggtgcagtatgcagggacagatggaccctgcaagatcccagtccagatggcggtggacatgcagaccctgaccccagaggaaggctgataaccgccaaccccgtgattactgaaagcactgagaactcaaagatgatgaggagcttgacccaccatttggggattcttacattgtcataggagttggggacaagaaaatcacccaccactggcataggagtggtagcaccatcggaaaggcatttgaggccactgtgagaggcgccaagagaatggcagtcctgggggatacagcctgggacttcggatcagtcgggggtgtgttcaactcactgggtaagggcattcaccagatttttggagcagccttcaaatcactgtttggaggaatgtcctggttctcacagatcctcataggcacgctgctagtgtggttaggtttgaacacaaagaatggatctatctccctcacatgcttggccctggggggagtgatgatcttcctctccacggctgtttctgctgacgtggggtgctcagtggacttctcaaaaaaggaaacgagatgtggcacgggggtattcatctataatgatgttgaagcctggagggaccggtacaagtaccatcctgactccccccgcagattggcagcagcagtcaagcaggcctgggaagaggggatctgtgggatctcatccgtttcaagaatggaaaacatcatgtggaaatcagtagaaggggagctcaatgctatcctagaggagaatggagttcaactgacagttgagtgggatctgtaaaaaaccccatgtggagaggtccacaaagattgccagtgcctgtgaatgagctgccccatggctggaaagcctgggggaaatcgtattttgttagggcggcaaagaccaacaacagttttgttgtcgacggtgacacactgaaggaatgtccgcttgagcacagagcatggaatagttttcttgtggaggatcacgggtttggagtcttccacaccagtgtctggcttaaggtcagagaagattactcattagaatgtgacccagccgtcataggaacagctgttaagggaagggaggccgcgcacagtgatctgggctattggattgaaagtgaaaagaatgacacatggaggctgaagagggcccacctgattgagatgaaaacatgtgaatggccaaagtctcacacattgtggacagatggagtagaagaaagtgatcttatcatacccaagtctttagctggtccactcagccaccacaacaccagagagggttacagaacccaagtgaaagggccatggcacagtgaagagcttgaaatccggtttgaggaatgtccaggcaccaaggtttacgtggaggagacatgcggaactagaggaccatctctgagatcaactactgcaagtggaagggtcattgaggaatggtgctgtagggaatgcacaatgcccccactatcgtttcgagcaaaagacggctgctggtatggaatggagataaggcccaggaaagaaccagagagcaacttagtgaggtcaatggtgacagcggggtcaaccgatcatatggaccacttctctcttggagtgcttgtgattctactcatggtgcaggaggggttgaagaagagaatgaccacaaagatcatcatgagcacatcaatggcagtgctggtagtcatgatcttgggaggattttcaatgagtgacctggccaagcttgtgatcctgatgggtgctactttcgcagaaatgaacactggaggagatgtagctcacttggcattggtagcggcatttaaagtcagaccagccttgctggtctccttcattttcagagccaattggacaccccgtgagagcatgctgctagccctggcttcgtgtcactgcaaactgcgatctctgctcttgaaggtgacttgatggtcctcattaatggatttgctaggcctggttggcaattcgagcaatggccgtgccacgcactgacaacatcgctctaccaatcaggctgctctaacaccactagctcgaggcacactgctcgtggcatggagagcgggcctggctacttgtggagggatcatgctcctctccctgaaagggaaaggtagtgtgaagaagaacctgccatttgtcatggccctgggattgacagctgtgagggtagtagaccctattaatgtggtaggactactgttactcacaaggagtgggaagcggagctggccccctagtgaagttctcacagccgttggcctgatatgtgcactggccggagggtttgccaaggcagacattgagatggctggacccatggctgcagtaggcttgctaattgtcagctatgtggtctcgggaaagagtgtggacatgtacattgaaagagcaggtgacatcacatgggaaaaggacgcggaagtcactggaaacagtcctcggcttgacgtggcactggatgagagtggtgacttctccttggtagaggaagatggtccacccatgagagagatcatactcaaggtggtcctgatggccatctgtggcatgaacccaatagctataccttttgctgcaggagcgtggtatgtgtatgtgaagactgggaaaaggagtggcgccctctgggacgtgcctgctcccaaagaagtgaagaaaggagagaccacagatggagtgtacagagtgatgactcgcagactgctaggttcaacacaggttggagtgggagtcatgcaagagggagtcttccacaccatgtggcacgttacaaaaggagccgcactgaggagcggtgagggaagacttgatccatactggggggatgtcaagcaggacttggtgtcatactgtgggccttggaagttggatgcagcttgggatggactcagcgaggtacagcttaggccgtacctcccggagagagggccagaaacattcagaccctgcctggaatattcaagacaaaggacggggacatcggagcagttgctctggactaccctgcagggacctcaggatctccgatcctagacaaatgtggaagagtgataggactctatggcaatggggagtgatcaagaatggaagctatgttagtgctataacccagggaaagagggaggaggagactccggttgaatgtttcgaaccctcgatgctgaagaagaagcagctaactgtcttggatctgcatccaggagccggaaaaaccaggagagttcttcctgaaatagtccgtgaagccataaaaaagagactccggacagtgatcaggcaccaactagggttgtcgctgctgagatggaggaggccttgagaggacttccggtgcgttacatgacaacagcagtcaacgtcacccattctgggacagaaatcgttgatttgatgtgccatgccactttcacttcacgcttactacaacccatcagagtccctaattacaatctcaacatcatggatgaagcccacttcacagacccctcaagtatagctgcaagaggatacatatcaacaagggagaaatgggcgaggcggctgccatttttatgactgccacaccaccaggaacccgtgatgcgtttcctgactctaactcaccaatcatggacacagaagtggaagtcccagagagagcctggagctcaggctttgattgggtgacagaccattctgggaaaacagtttggttcgttccaagcgtgagaaacggaaatgaaatcgcagcctgtctgacaaaggctggaaagcgggtcatacagctcagcaggaagacttttgagacagaatttcagaaaacaaaaaatcaagagtgggactttgtcataacaactgacatctcagagatgggcgccaacttcaaggctgaccgggtcatagactctaggagatgcctaaaaccagtcatacttgatggtgagagagtcatcttggctgggcccatgcctgtcacgcatgctagtgctgctcagaggagaggacgtataggcaggaaccctaacaaacctggagatgagtacatgtatggaggtgggtgtgcagagactgatgaaggccatgcacactggcttgaagcaagaatgcttcttgacaacatctacctccaggatggcctcatagcctcgctctatcggcctgaggccgataaggtagccgccattgagggagagtttaagctgaggacagagcaaaggaagaccttcgtggaactcatgaagagaggagaccttcccgtctggctagcctatcaggttgcatctgccggaataacttacacagacagaagatggtgctttgatggcacaaccaacaacaccataatggaagacagtgtaccagcagaggtttggacaaagtatggagagaagagagtgctcaaaccgagatggatggatgctagggtctgttcagaccatgcggccctgaagtcgttcaaagaattcgccgctggaaaaagaggagcggctttgggagtaatggaggccctgggaacactgccaggacacatgacagagaggtttcaggaagccattgacaacctcgccgtgctcatgcgagcagagactggaagcaggccttataaggcagcggcagcccaactgccggagaccctagagaccattatgctcttaggtttgctgggaacagtttcactggggatcttcacgtcttgatgcggaataagggcatcgggaagatgggctttggaatggtaacccttggggccagtgcatggctcatgtggctacggaaattgaaccagccagaattgcatgtgtcctcattgagtgatttattactggtggtgctcatacccgagccagagaagcaaagatctccccaagataaccagatggcaattatcatcatggtggcagtgggccttctaggtttgataactgcaaacgaacttggatggctggaaagaacaaaaaatgacatagctcatctaatgggaaggagagaagaaggagcaaccatgggattctcaatggacattgatctgcggccagcctccgcctgggctatctatgccgcattgacaactctcatcaccccagctgtccaacatgcggtaaccacttcatacaacaactactccttaatggcgatggccacacaagctggagtgctgtaggcatgggcaaagggatgccatttatgcatggggaccttggagtcccgctgctaatgatgggttgctattcacaattaacacccctgactctgatagtagctatcattctgcttgtggcgcactacatgtacttgatcccaggcctacaagcggcagcagcgcgtgctgcccagaaaaggacagcagctggcatcatgaagaatcccgagtggatggaatagtggtaactgacattgacacaatgacaatagacccccaggtggagaagaagatgggacaagtgttactcatagcagtagccatctccagtgctgtgctgctgcggaccgcctggggatggggggaggctggagctctgatcacagcagcgacctccaccttgtgggaaggctctccaaacaaatactggaactcctctacagccacctcactgtgcaacatcttcagaggaagctatctggcaggagcttcccttatctatacagtgacgagaaacgctggcctggttaagagacgtggaggtgggacgggagagactctgggagagaagtggaaagctcgtctgaatcagatgtcggccctggagttctactcttataaaaagtcaggtatcactgaagtgtgtagagaggaggctcgccgtgccctcaaggatggagtggccacaggaggacatgccgtatcccggggaagtgcaaagatcagatggttggaggagagaggatatctgcagccctatgggaaggttgttgacctcggatgtggcagagggggctggagctattatgccgccaccatccgcaaagtgcaggaggtgagaggatacacaaagggaggtcccggtcatgaagaacccatgctggtgcaaagctatgggtggaacatagttcgtctcaagagtggagtggacgtcttccacatggcggctgagccgtgtgacactctgctgtgtgacataggtgagtcatcatctagtcctgaagtggaagagacacgaacactcagagtgctctctatggtgggggactggcttgaaaaaagaccaggggccttctgtataaaggtgctgtgcccatacaccagcactatgatggaaaccatggagcgactgcaacgtaggcatgggggaggattagtcagagtgccattgtgtcgcaactccacacatgagatgtactgggtctctggggcaaagagcaacatcataaaaagtgtgtccaccacaagtcagctcctcctgggacgcatggatggccccaggaggccagtgaaatatgaggaggatgtgaacctcggctcgggtacacgagctgtggcaagctgtgctgaggctcctaacatgaaaatcatcggcaggcgcattgagagaatccgcaatgaacatgcagaaacatggtttcttgatgaaaaccacccatacaggacatgggcctaccatgggagctacgaagcccccacgcaaggatcagcgtcttccctcgtgaacggggttgttagactcctgtcaaagccttgggacgtggtgactggagttacaggaatagccatgactgacaccacaccatacggccaacaaagagtcttcaaagaaaaagtggacaccagggtgccagatccccaagaaggcactcgccaggtaatgaacatagtctcttcctggctgtggaaggagctggggaaacgcaagcggccacgcgtctgcaccaaagaagagtttatcaacaaggtgcgcagcaatgcagcactgggagcaatatttgaagaggaaaaagaatggaagacggctgtggaagctgtgaatgatccaaggttttgggccctagtggatagggagagagaacaccacctgagaggagagtgtcacagctgtgtgtacaacatgatgggaaaaagagaaaagaagcaaggagagttcgggaaagcaaaaggtagccgcgccatctggtacatgtggttgggagccagattcttggagatgaagcccttggattcttgaacgaggaccattggatgggaagagaaaactcaggaggtggagtcgaagggttaggattgcaaagacttggatacattctagaagaaatgaatcgggcaccaggaggaaagatgtacgcagatgacactgctggctgggacacccgcattagtaagtttgatctggagaatgaagctctgattaccaaccaaatggaggaagggcacagaactctggcgttggccgtgattaaatacacataccaaaacaaagtggtgaaggttctcagaccagctgaaggaggaaaaacagttatggacatcatttcaagacaagaccagagagggagtggacaagttgtcacttatgctctcaacacattcaccaacttggtggtgcagcttatccggaacatggaagctgaggaagtgttagagatgcaagacttatggttgttgaggaagccagagaaagtgaccagatggttgcagagcaatggatgggatagactcaaacgaatggcggtcagtggagatgactgcgttgtgaagccaatcgatgataggtttgcacatgccctcaggttcttgaatgacatgggaaaagttaggaaagacacacaggagtggaaaccctcgactggatggagcaattgggaagaagtcccgttctgctcccaccacttcaacaagctgtacctcaaggatgggagatccattgtggtcccttgccgccaccaagatgaactgattggccgagctcgcgtctcaccaggggcaggatggagcatccgggagactgcctgtcttgcaaaatcatatgcgcagatgtggcagctcctttatttccacagaagagaccttcgactgatggctaatgccatttgctcggctgtgccagttgactgggtaccaactgggagaaccacctggtcaatccatggaaagggagaatggatgaccactgaggacatgctcatggtgtggaatagagtgtggattgaggagaacgaccatatggaggacaagactcctgtaacaaaatggacagacattccctatctaggaaaaagggaggacttatggtgtggatcccttatagggcacagaccccgcaccacttgggctgaaaacatcaaagacacagtcaacatggtgcgcaggatcataggtgatgaagaaaagtacatggactatctatccacccaagtccgctacttgggtgaggaagggtccacacccggagtgttgtaagcaccaattttagtgagtcaggcctgctagtcagccacagatggggaaagctgtgcagcctgtaacccccccaggagaagctgggaaaccaagctcatagtcaggccgagaacgccatggcacggaagaagccatgctgcctgtgagcccctcagaggacactgagtcaaaaaaccccacgcgcaggaagcgcaggatgggaaaagaaggtggcgaccttccccacccttcaatctggggcctgaactggagactagctgtgaatctccagcagagggactagtggttagaggagaccccccggaaaacgcaaaacagcatattgacgtgggaaagaccagagactccatgagtttccaccacgctggccgccaggcacagatcgccgaacttcggcggccggtg tggggaaatccatggtttct

In certain aspects of the disclosures herein, a combination of at leasttwo oligomers is provided for determining the presence or absence ofZika virus in a sample. Typically, the oligomer combination includes atleast two amplification oligomers for amplifying a target sequence of aZika virus target nucleic acid corresponding to the sequence within SEQID NO:1. In such embodiments, at least one amplification oligomercomprises a target-hybridizing sequence in the sense orientation and atleast one amplification oligomer comprises a target-hybridizing sequencein the antisense orientation, where the amplification oligomers are eachconfigured to specifically hybridize to a Zika virus target sequencecorresponding to a sequence contained within SEQ ID NO:1 and where thetarget-hybridizing sequences are selected so that the amplificationoligomers are situated to hybridize the ends of the target sequence tobe amplified. In some variations, the at least two amplificationoligomers are configured to specifically hybridize to a target sequencein one or more of the following nucleic acid sequences SEQ ID NO:183,SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ IDNO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:194, thereverse-complement of any one of SEQ ID NO:183, SEQ ID NO:184, SEQ IDNO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQID NO:190, SEQ ID NO:194, an RNA equivalents of any one of SEQ IDNO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:194, andreverse-complements of an RNA equivalent of SEQ ID NO:183, SEQ IDNO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQID NO:189, SEQ ID NO:190, SEQ ID NO:194. In some variations, the atleast two amplification oligomers are configured to specificallyhybridize to a target sequence consisting essentially of: SEQ ID NO:183,SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ IDNO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:194, thereverse-complement of any one of SEQ ID NO:183, SEQ ID NO:184, SEQ IDNO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQID NO:190, SEQ ID NO:194, an RNA equivalents of any one of SEQ IDNO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:194, orreverse-complements of an RNA equivalent of SEQ ID NO:183, SEQ IDNO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQID NO:189, SEQ ID NO:190, SEQ ID NO:194.

TABLE 1 Exemplary Amplification Oligomer Target-hybridizingSequences for Amplification of Zika virus Target Sequences SEQ ID NO:Sequence (5′ to 3′)   2 TCYCTTGGAGTGCTTGTGA   3 TCYCTTGGAGTGCTTGTGATT  4 TCYCTTGGAGTGCTTGTGATTYT   5 TCYCTTGGAGTGCTTGTGATTY   6YCCYAAYAAACCTGGAGATGAGTA   7 AAYAAACCTGGAGATG   8 YAAYAAACCTGGAGATG   9TGGCTTGAAGCAAGAATGCT  10 GCTTGAAGCAAGAAT  11 AGGACAGCAGCTGGCATCAT  12AGGACGGCAGCTGGCATCAT  13 AGGACAGCAGCTGGCATCATG  14 AGGACGGCAGCTGGCATCATG 15 AGGACAGCAGCTGGCATCATGA  16 AGGACGGCAGCTGGCATCATGA  17AGGACAGCAGCTGGCATCATGAA  18 AGGACGGCAGCTGGCATCATGAA  19ACGGCAGCTGGCATCATGAA  20 GACGGCAGCTGGCATCATGAA  21ACAGCAGCTGGCATCATGAAGAA  22 AGGACAGCAGCTGGCATCATGAAGAA  23AGAACAGCAGCTGGCATCATGAAGAA  24 AGRACRGCAGCTGGCATCATGAAGAA  25ACRGCAGCTGGCATCAT  26 RACRGCAGCTGGCATCATGAA  27 GTTGTGGATGGAATAGTGGT  28TCTCTTGGAGTGCTTGTGATTC  29 TCTCTTGGAGTGCTTGTGATT  30 TCTCTTGGAGTGCTTGTGA 31 TCTCTTGGAGTGCTTGTGATTCT  32 TCCCTTGGAGTGCTTGTGATTCT  33TCCCTTGGAGTGCTTGTGATTC  34 TCCCTTGGAGTGCTTGTGATT  35 TCCCTTGGAGTGCTTGTGA 36 AACAAACCTGGAGATGAGTA  37 CAACAAACCTGGAGATGAGTA  38AACAAACCTGGAGATGAGT  39 CAACAAACCTGGAGATGAGT  40 CAATAAACCTGGAGATGAGT 41 CCCAATAAACCTGGAGATGAGT  42 CCYAAYAAACCTGGAGATGAGTA  43CCCAACAAACCTGGAGATGAGTA  44 YCCYAAYAAACCTGGAGATG  45TCCTAACAAACCTGGAGATG  46 CCCAACAAACCTGGAGATGAGT  47CCYAAYAAACCTGGAGATGAG  48 CCCAACAAACCTGGAGATGAG  49AGRACRGCAGCTGGCATCATGAAGA  50 AGRACRGCAGCTGGCATCATGAAGAA  51AGRACRGCAGCTGGCATCATGA  52 AGRACRGCAGCTGGCATCAT  53AGRACRGCAGCTGGCATCATGAA 111 TRRCTACCAGCACTGCCAT 112 GTCWATWGTCATTGTGT113 GTCATTGTGTCAATGTCAG 114 GTCTATTGTCATTGTGT 115CTGCTATGAGTAACACTTGTCCCATCTT 116 TAGCTACCAGCACTGCCAT 117CTACCAGCACTGCCATTGATGTGC 118 CTGCYATGAGTARCACYTGYCCCATCTT 119GTCWATWGTCATTGTGTCA 120 YTGYCCCATCTTYTTYT 121 GTCWATWGTCATTGTGTCAATGTCAG122 TRRCTACCAGCACTGCCATTG 123 GRAGCATTCTTGCTTCAAGCCA 124GTCAAGRAGCATTCTTGCTTCA 125 GTCATTGTGTCAATGTCAGT 126 TCATTGTGTCAATGTCAGT127 GTCTATTGTCATTGTGTCA 128 ATTGTCATTGTGTCAATGTCAGT 129ATTGTCATTGTGTCAATGTCA 130 ATTGTCATTGTGTCAATGTC 131 ATTGTCATTGTGTCAATGT132 TTGTCCCATCTTCTTCT 133 GTAACACTTGTCCCATCTT 134GTCTATTGTCATTGTGTCAATGTCAG 135 CTACCAGCACTGCCATTGATGTGCT 136CCAGCACTGCCATTGATGTGCT 137 CTACCAGCACTGCCATTGATGT 138TAGCTACCAGCACTGCCATTG 139 AGCATTCTTGCTTCAAGCCA 140 GCATTCTTGCTTCAAGCCA141 GGAGCATTCTTGCTTCAAGCCA 142 GTCAAGAAGCATTCTTGCTTCA 143TAGAGCGAGGCTATGAGGCCATC 144 TAGAGCGAGGCTATGAGGCCAT 145TAGAGCGAGGCTATGAGGCCA 146 TAGAGCGAGGCTATGAGG 147 TAGAGCGAGGCTATGAG

In certain embodiments, an amplification oligomer as described herein isa promoter-based amplification oligomer comprising a target-hybridizingsequence and further comprising a promoter sequence located 5′ to thetarget-hybridizing sequence wherein the promoter sequence isnon-complementary to the Zika virus target nucleic acid. For example, insome embodiments of an oligomer combination as described herein foramplification of a Zika virus target sequence, an amplification oligomeras described above in Table 1 is a promoter-based amplificationoligomer, wherein a target-hybridizing sequence further comprises a 5′promoter sequence. In particular embodiments, the promoter sequence is aT7 RNA polymerase promoter sequence such as, for example, a T7 promotersequence having the sequence shown in SEQ ID NO:179. In specificvariations, the amplification oligomer is a has the nucleotide sequenceshown in SEQ ID NOs:74-110.

In some embodiments, an oligomer combination as described herein furthercomprises at least one capture probe oligomer comprising atarget-hybridizing sequence substantially corresponding to a sequencecontained in the complement of SEQ ID NO:1, wherein thetarget-hybridizing sequence is covalently attached to a sequence ormoiety that binds to an immobilized probe. In specific variations, thetarget-hybridizing sequence comprises or consists of a sequencesubstantially corresponding to, or identical to, a sequence selectedfrom SEQ ID NOs:166-178, including complements, DNA equivalents, andDNA/RNA chimerics thereof. In more specific variations, the captureprobe oligomer has a sequence selected from SEQ ID NOs:153-165. Anoligomer combination may include at least two capture probe oligomers asabove. In more specific embodiments, the at least one capture probeoligomer includes providing the at least one capture probe oligomer in atarget capture reaction mixture. In one aspect, each of the at least onecapture probe oligomers is provided in the target capture reactionmixture at a concentration from about 3 pmoles/reaction to about 6pmoles/reaction (inclusive of all whole and partial numbers of the range(e.g., 4, 4.75, 5.12, 5.98, 6)). When a plurality of at least onecapture probe oligomer is used in a target capture reaction theconcentration of each capture probe oligomer may be equal to theconcentration of the others or there may be varied concentrations, asdescribed herein.

In certain variations, an oligomer combination as described hereinfurther comprises at least one detection probe oligomer configured tospecifically hybridize to a Zika virus target sequence that isamplifiable using the first and second amplification oligomers (e.g., aZika virus target sequence that is flanked by the target-hybridizingsequences of the first and second amplification oligomers). Particularlysuitable detection probe oligomers include, for example, oligomerscomprising a target-hybridizing sequence substantially corresponding to,or identical to, a sequence selected from SEQ ID NOs:54-63 & 66-73,including complements, DNA equivalents, and DNA/RNA chimerics thereof. Adetection probe oligomer may contain a 2′-methoxy backbone at one ormore linkages in the nucleic acid backbone. In some variations, anoligomer combination includes at least two detection probe oligomers. Inmore specific embodiments, the at least one detection probe oligomerincludes providing the at least one detection probe oligomer in anamplicon detection reaction mixture. In one aspect, each of the at leastone detection probe oligomers is provided in the detection reactionmixture at about 2.0 E+06 RLU/reaction to about 6.0 E+06 RLU/reaction(inclusive of all whole and partial numbers of the range (e.g., 2.0E+06, 2.138 E+06, 3.385 E+06 RLU)). When a plurality of at least onedetection probe oligomer is used in a detection reaction theconcentration of each detection oligomer may be equal to theconcentration of the others or there may be varied concentrations, asdescribed herein.

Typically, a detection probe oligomer further includes a label.Particularly suitable labels include compounds that emit a detectablelight signal, e.g., fluorophores or luminescent (e.g., chemiluminescent)compounds that can be detected in a homogeneous mixture. More than onelabel, and more than one type of label, may be present on a particularprobe, or detection may rely on using a mixture of probes in which eachprobe is labeled with a compound that produces a detectable signal (see,e.g., U.S. Pat. Nos. 6,180,340 and 6,350,579). Labels may be attached toa probe by various means including covalent linkages, chelation, andionic interactions, but preferably the label is covalently attached. Forexample, in some embodiments, a detection probe has an attachedchemiluminescent label such as, e.g., an acridinium ester (AE) compound(see, e.g., U.S. Pat. Nos. 5,185,439; 5,639,604; 5,585,481; and5,656,744), which in typical variations is attached to the probe by anon-nucleotide linker (see, e.g., U.S. Pat. Nos. 5,585,481; 5,656,744;and 5,639,604, particularly at column 10, line 6 to column 11, line 3,and Example 8). In other embodiments, a detection probe comprises both afluorescent label and a quencher, a combination that is particularlyuseful in fluorescence resonance energy transfer (FRET) assays. Specificvariations of such detection probes include, e.g., a TaqMan detectionprobe (Roche Molecular Diagnostics), a molecular torch (see, e.g., U.S.Pat. Nos. 6,849,412; 6,835,542; 6,534,274; and 6,361,945), and a“molecular beacon” (see, e.g., Tyagi et al., Nature Biotechnol.16:49-53, 1998; U.S. Pat. Nos. 5,118,801 and 5,312,728). In someembodiments, the detections probe oligomers are molecular torcholigomers comprising a target-hybridizing sequence selected from SEQ IDNOs:54-63 & 66-73, including complements, DNA equivalents, and DNA/RNAchimerics thereof.

In another aspect, the present invention provides methods fordetermining the presence or absence of Zika virus in a sample using anoligomer combination as described herein. Such a method generallyincludes (1) contacting the sample with at least two oligomers foramplifying a Zika virus nucleic acid target sequence corresponding to aZika virus target nucleic acid, where the oligomers include at least twoamplification oligomers as described herein; (2) performing an in vitronucleic acid amplification reaction, where any Zika virus target nucleicacid present in the sample is used as a template for generating anamplification product; and (3) detecting the presence or absence of theamplification product, thereby determining the presence or absence ofZika virus in the sample. A detection method in accordance with thepresent invention typically further includes the step of obtaining thesample to be contacted with the at least two oligomers. In certainembodiments, “obtaining” a sample to be used in steps (1)-(3) includes,for example, receiving the sample at a testing facility or otherlocation where one or more steps of the method are performed, and/orretrieving the sample from a location (e.g., from storage or otherdepository) within a facility where one or more steps of the method areperformed. In certain embodiments, the sample is contacted with at leasttwo amplification oligomers for amplifying a Zika virus nucleic acidtarget sequence corresponding to a Zika virus target nucleic acid, wherethe oligomers include at least two amplification oligomers as describedabove; (2) performing an in vitro nucleic acid amplification reaction,where any Zika virus target nucleic acid present in the sample is usedas a template for generating an amplification product and theamplification product sequence substantially corresponds to, or consistsessentially of, or is identical to one or more of the followingsequences SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186,SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ IDNO:194 SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, thereverse-complement of any one of SEQ ID NO:183, SEQ ID NO:184, SEQ IDNO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQID NO:190, SEQ ID NO:194 SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197,SEQ ID NO:198, an RNA equivalents of any one of SEQ ID NO:183, SEQ IDNO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQID NO:189, SEQ ID NO:190, SEQ ID NO:194 SEQ ID NO:195, SEQ ID NO:196,SEQ ID NO:197, SEQ ID NO:198, and reverse-complements of an RNAequivalent of SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ IDNO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQID NO:194 SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198. Incertain embodiments, an amplification product is contacted with adetection probe oligomer that is configured to specifically hybridize toa target sequence in one or more of the following sequences SEQ IDNO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:194 SEQ ID NO:195,SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, the reverse-complement ofany one of SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186,SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ IDNO:194 SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, anRNA equivalents of any one of SEQ ID NO:183, SEQ ID NO:184, SEQ IDNO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQID NO:190, SEQ ID NO:194 SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197,SEQ ID NO:198, and reverse-complements of an RNA equivalent of SEQ IDNO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:194 SEQ ID NO:195,SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198.

In certain embodiments, the method further includes purifying the Zikavirus target nucleic acid from other components in the sample before thecontacting step. Such purification may include methods of separatingand/or concentrating organisms contained in a sample from other samplecomponents. In particular embodiments, purifying the target nucleic acidincludes capturing the target nucleic acid to specifically ornon-specifically separate the target nucleic acid from other samplecomponents. Non-specific target capture methods may involve selectiveprecipitation of nucleic acids from a substantially aqueous mixture,adherence of nucleic acids to a support that is washed to remove othersample components, or other means of physically separating nucleic acidsfrom a mixture that contains Zika virus nucleic acid and other samplecomponents.

In some embodiments, a Zika virus target nucleic is selectivelyseparated or purified away from other sample components by specificallyhybridizing the Zika virus target nucleic acid to a capture probeoligomer. The capture probe oligomer comprises a target-hybridizingsequence configured to specifically hybridize to a Zika virus targetsequence so as to form a target-sequence:capture-probe complex that isseparated from sample components. Suitable capture probetarget-hybridizing sequences include sequences substantiallycorresponding to, or identical to, a sequence selected from SEQ IDNOs:166-178, including complements, DNA equivalents, and DNA/RNAchimerics thereof. In a preferred variation, the specific target capturebinds the Zika virus target:capture-probe complex to an immobilizedprobe to form a target:capture-probe-immobilized-probe complex that isseparated from the sample and, optionally, washed to remove non-targetsample components (see, e.g., U.S. Pat. Nos. 6,110,678; 6,280,952; and6,534,273). In such variations, the capture probe oligomer furthercomprises a moiety that binds attaches the capture probe, with its boundtarget sequence, to an immobilized probe attached to a solid support,thereby permitting the hybridized target nucleic acid to be separatedfrom other sample components. In a preferred embodiment, the immobilizedprobe is a substantially homopolymeric sequence, in a more preferredembodiment, the immobilized probe is a poly-T sequence. By way ofexample only, in a preferred embodiment, the immobilized probe is a dT₁₄sequence (SEQ ID NO:180). By way of example only, in a preferredembodiment, the capture probe comprises a moiety that is a nucleic acidsequence, preferably SEQ ID NO:181 or SEQ ID NO:182.

In more specific embodiments, the capture probe oligomer includes a tailportion (e.g., a 3′ tail) that is a nucleic acid sequence, wherein thenucleic acid sequence is not complementary to the Zika virus targetsequence but that specifically hybridizes to a sequence on theimmobilized probe, thereby serving as the moiety allowing the targetnucleic acid to be separated from other sample components, such aspreviously described in, e.g., U.S. Pat. No. 6,110,678, incorporatedherein by reference. Any sequence may be used in a tail region, which isgenerally about 5 to 50 nt long, and preferred embodiments include asubstantially homopolymeric tail of about 10 to 40 nt (e.g., A₁₀ toA₄₀), more preferably about 14 to 33 nt (e.g., A₁₄ to A₃₀ or T₃A₁₄ toT₃A₃₀), that bind to a complementary immobilized sequence (e.g., poly-T)attached to a solid support, e.g., a matrix or particle. By way ofexample only, in a preferred embodiment the substantially homopolymerictail is SEQ ID NO181 or 182 and the immobilized probe is SEQ ID NO:180.For example, in specific embodiments of a capture probe comprising a 3′tail, the capture probe has a sequence selected from SEQ ID NOs:153-165.

Target capture typically occurs in a solution phase mixture thatcontains one or more capture probe oligomers that hybridize specificallyto the Zika virus target sequence under hybridizing conditions, usuallyat a temperature higher than the T. of thetail-sequence-immobilized-probe-sequence duplex. For embodimentscomprising a capture probe tail, the Zika virus-target:capture-probecomplex is captured by adjusting the hybridization conditions so thatthe capture probe tail hybridizes to the immobilized probe, and theentire complex on the solid support is then separated from other samplecomponents. The support with the attachedimmobilized-probe:capture-probe:Zika virus-target-sequence may be washedone or more times to further remove other sample components. Preferredembodiments use a particulate solid support, such as paramagnetic beads,so that particles with the attached Zikavirus-target:capture-probe-immobilized-probe complex may be suspended ina washing solution and retrieved from the washing solution, preferablyby using magnetic attraction. To limit the number of handling steps, theZika virus target nucleic acid may be amplified by simply mixing theZika virus target sequence in the complex on the support withamplification oligomers and proceeding with amplification steps.

Amplifying a Zika virus target sequence utilizes an in vitroamplification reaction using at least two amplification oligomers thathybridize a target sequence to be amplified. In particular embodiments,the target sequence to be amplified substantially corresponds to all ora portion of SEQ ID NO:1 from about nucleotide position 3340 to aboutnucleotide position 3862, or from about nucleotide position 5806 toabout nucleotide position 6380, or from about nucleotide position 7132to about nucleotide position 7641, or a combination thereof. Inparticular embodiments, the target sequence is contained within asequence from about nucleotide position 3340 to about nucleotideposition 3862 of SEQ ID NO:1, or from about nucleotide position 5806 toabout nucleotide position 6380 or SEQ ID NO:1, or from about nucleotideposition 7132 to about nucleotide position 7641 of SEQ ID NO:1. Inparticular embodiments, a target sequence is contained within SEQ IDNO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:194, thereverse-complement of any one of SEQ ID NO:183, SEQ ID NO:184, SEQ IDNO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQID NO:190, SEQ ID NO:194, an RNA equivalents of any one of SEQ IDNO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:194, orreverse-complements of an RNA equivalent of SEQ ID NO:183, SEQ IDNO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQID NO:189, SEQ ID NO:190, SEQ ID NO:194. In particular embodiments, atarget sequence is contained within a sequence consisting essentially ofSEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ IDNO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:194, thereverse-complement of any one of SEQ ID NO:183, SEQ ID NO:184, SEQ IDNO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQID NO:190, SEQ ID NO:194, an RNA equivalents of any one of SEQ IDNO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:194, orreverse-complements of an RNA equivalent of SEQ ID NO:183, SEQ IDNO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQID NO:189, SEQ ID NO:190, SEQ ID NO:194. Particularly suitableamplification oligomer combinations for amplification of these Zikavirus regions are described herein. Suitable amplification methodsinclude, for example, replicase-mediated amplification, polymerase chainreaction (PCR), ligase chain reaction (LCR), strand-displacementamplification (SDA), and transcription-mediated ortranscription-associated amplification (TMA). Such amplification methodsare well-known in the art and are readily used in accordance with themethods of the present invention.

For example, some amplification methods that use TMA amplificationinclude the following steps. Briefly, the target nucleic acid thatcontains the sequence to be amplified is provided as single-strandednucleic acid (e.g., ssRNA or ssDNA). Those skilled in the art willappreciate that conventional melting of double stranded nucleic acid(e.g., dsDNA) may be used to provide single-stranded target nucleicacids. A promoter-based amplification oligomer binds specifically to thetarget nucleic acid at its target sequence and a reverse transcriptase(RT) extends the 3′ end of the promoter primer using the target strandas a template to create a cDNA copy of the target sequence strand,resulting in an RNA:DNA duplex. An RNase digests the RNA strand of theRNA:DNA duplex and a second primer binds specifically to its targetsequence, which is located on the cDNA strand downstream from thepromoter primer end. RT synthesizes a new DNA strand by extending the 3′end of the second primer using the first cDNA template to create a dsDNAthat contains a functional promoter sequence. An RNA polymerase specificfor the promoter sequence then initiates transcription to produce RNAtranscripts that are about 100 to 1000 amplified copies (“amplicons”) ofthe initial target strand in the reaction. Amplification continues whenthe second primer binds specifically to its target sequence in each ofthe amplicons and RT creates a DNA copy from the amplicon RNA templateto produce an RNA:DNA duplex. RNase in the reaction mixture digests theamplicon RNA from the RNA:DNA duplex and the promoter primer bindsspecifically to its complementary sequence in the newly synthesized DNA.RT extends the 3′ end of the promoter primer to create a dsDNA thatcontains a functional promoter to which the RNA polymerase binds totranscribe additional amplicons that are complementary to the targetstrand. The autocatalytic cycles of making more amplicon copies repeatduring the course of the reaction resulting in about a billion-foldamplification of the target nucleic acid present in the sample. Theamplified products may be detected in real-time during amplification, orat the end of the amplification reaction by using a probe that bindsspecifically to a target sequence contained in the amplified products.Detection of a signal resulting from the bound probes indicates thepresence of the target nucleic acid in the sample.

In some embodiments, TMA methods utilize a “reverse” TMA reaction. Insuch variations, the initial or “forward” amplification oligomer is apriming oligonucleotide that hybridizes to the target nucleic acid inthe vicinity of the 3′-end of the target sequence. A reversetranscriptase (RT) synthesizes a cDNA strand by extending the 3′-end ofthe primer using the target nucleic acid as a template. The second or“reverse” amplification oligomer is a promoter primer or promoterprovider having a target-hybridizing sequence configured to hybridize toa target-sequence contained within the synthesized cDNA strand. Wherethe second amplification oligomer is a promoter primer, RT extends the3′ end of the promoter primer using the cDNA strand as a template tocreate a second, cDNA copy of the target sequence strand, therebycreating a dsDNA that contains a functional promoter sequence.Amplification then continues essentially as described above forinitiation of transcription from the promoter sequence utilizing an RNApolymerase. Alternatively, where the second amplification oligomer is apromoter provider, a terminating oligonucleotide, which hybridizes to atarget sequence that is in the vicinity to the 5′-end of the targetsequence, is typically utilized to terminate extension of the primingoligomer at the 3′-end of the terminating oligonucleotide, therebyproviding a defined 3′-end for the initial cDNA strand synthesized byextension from the priming oligomer. The target-hybridizing sequence ofthe promoter provider then hybridizes to the defined 3′-end of theinitial cDNA strand, and the 3′-end of the cDNA strand is extended toadd sequence complementary to the promoter sequence of the promoterprovider, resulting in the formation of a double-stranded promotersequence. The initial cDNA strand is then used a template to transcribemultiple RNA transcripts complementary to the initial cDNA strand, notincluding the promoter portion, using an RNA polymerase that recognizesthe double-stranded promoter and initiates transcription therefrom. Eachof these RNA transcripts is then available to serve as a template forfurther amplification from the first priming amplification oligomer.

Detection of the amplified products may be accomplished by a variety ofmethods. The nucleic acids may be associated with a surface that resultsin a physical change, such as a detectable electrical change. Amplifiednucleic acids may be detected by concentrating them in or on a matrixand detecting the nucleic acids or dyes associated with them (e.g., anintercalating agent such as ethidium bromide or cyber green), ordetecting an increase in dye associated with nucleic acid in solutionphase. Other methods of detection may use nucleic acid detection probesthat are configured to specifically hybridize to a sequence in theamplified product and detecting the presence of the probe:productcomplex, or by using a complex of probes that may amplify the detectablesignal associated with the amplified products (e.g., U.S. Pat. Nos.5,424,413; 5,451,503; and 5,849,481). Directly or indirectly labeledprobes that specifically associate with the amplified product provide adetectable signal that indicates the presence of the target nucleic acidin the sample. In particular, the amplified product will contain atarget sequence in or complementary to a sequence in the Zika virusgenomic RNA, and a probe will bind directly or indirectly to a sequencecontained in the amplified product to indicate the presence of Zikavirus nucleic acid in the tested sample.

Preferred embodiments of detection probes that hybridize to thecomplementary amplified sequences may be DNA or RNA oligomers, oroligomers that contain a combination of DNA and RNA nucleotides, oroligomers synthesized with a modified backbone, e.g., an oligomer thatincludes one or more 2′-methoxy substituted ribonucleotides. Probes usedfor detection of the amplified Zika virus sequences may be unlabeled anddetected indirectly (e.g., by binding of another binding partner to amoiety on the probe) or may be labeled with a variety of detectablelabels. Particular embodiments of detection probes suitable for use inaccordance with methods of the present invention are further describedherein. In some preferred embodiments of the method for detecting Zikavirus sequences, such as in certain embodiments usingtranscription-mediated amplification (TMA), the detection probe is alinear chemiluminescently labeled probe, more preferably, a linearacridinium ester (AE) labeled probe.

Other embodiments using transcription-mediated amplification utilize apromoter-based amplification oligomer, which comprises a firsttarget-hybridizing sequence and, situated 5′ to the first region, asecond region comprising a promoter sequence for an RNA polymerase, butwhich is not modified to prevent the initiation of DNA synthesis fromits 3′-terminus. In some embodiments, a promoter primer for use inaccordance with the detection method comprises a target-hybridizingsequence having a sequence substantially corresponding to, or identicalto, a sequence selected from SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76,SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81,SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86,SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91,SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96,SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101,SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ IDNO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, andSEQ ID NO:111.

Assays for detection of the Zika virus nucleic acid may optionallyinclude a non-Zika virus internal control (IC) nucleic acid that isamplified and detected in the same assay reaction mixtures by usingamplification and detection oligomers specific for the IC sequence. ICnucleic acid sequences can be RNA template sequences (e.g., and in vitrotranscript), synthetic nucleic acid sequences that are spiked into asample or the IC nucleic acid sequences may be a cellular component. ICnucleic acid sequences that are cellular components can be fromexogenous cellular sources or endogenous cellular sources relative tothe specimen. In these instances, an internal control nucleic acid isco-amplified with the Zika virus nucleic acid in the amplificationreaction mixtures. The internal control amplification product and theZika virus target sequence amplification product can be detectedindependently. Two different internal control systems were employed inthe procedures described below.

A first arrangement for internal control systems was useful formonitoring the integrity of amplification and detection reactions thatemploy paired sets of primers and an oligonucleotide probe thathybridized amplification product at a position between the primerbinding sites, or the complements thereof. In a simple application, theinternal control template nucleic acid can be distinguished from theanalyte template nucleic acid at the sequence of bases serving as theprobe binding site. These bases may be scrambled, replaced by anunrelated base sequence, or simply contain a sufficient number of pointmutations to result in differential probe binding. In this way, nucleicacid products resulting from amplification of analyte nucleic acid canbe detected by an analyte-specific probe, and not by an internalcontrol-specific probe. Likewise, amplicons resulting from amplificationof internal control nucleic acid can be detected by an internalcontrol-specific probe, and not by an analyte-specific probe. Thisconfiguration allows that both analyte and internal control nucleic acidtemplates may be amplified using identical primers, or primer sets.

In certain embodiments, amplification and detection of a signal from theamplified IC sequence demonstrates that the assay reagents, conditions,and performance of assay steps were properly used in the assay if nosignal is obtained for the intended target Zika virus nucleic acid(e.g., samples that test negative for Zika virus). An IC may also beused as an internal calibrator for the assay when a quantitative resultis desired, i.e., the signal obtained from the IC amplification anddetection is used to set a parameter used in an algorithm forquantitating the amount of Zika virus nucleic acid in a sample based onthe signal obtained for an amplified Zika virus target sequence. ICs arealso useful for monitoring the integrity of one or more steps in anassay. A preferred embodiment of a synthetic IC nucleic acid sequence isa randomized sequence that has been derived from a naturally occurringsource (e.g., an HIV sequence that has been rearranged in a randommanner). Another preferred IC nucleic acid sequence may be an RNAtranscript isolated from a naturally occurring source or synthesized invitro, such as by making transcripts from a cloned randomized sequencesuch that the number of copies of IC included in an assay may beaccurately determined. The primers and probe for the IC target sequenceare configured and synthesized by using any well-known method providedthat the primers and probe function for amplification of the IC targetsequence and detection of the amplified IC sequence using substantiallythe same assay conditions used to amplify and detect the Zika virustarget sequence. In preferred embodiments that include a targetcapture-based purification step, it is preferred that a target captureprobe specific for the IC target be included in the assay in the targetcapture step so that the IC is treated in the assay in a manneranalogous to that for the intended Zika virus analyte in all of theassay steps.

Also provided by the subject invention is a reaction mixture fordetermining the presence or absence of a Zika virus target nucleic acidin a sample. A reaction mixture in accordance with the present inventionat least comprises one or more of the following: an oligomer combinationas described herein for amplification of a Zika virus target nucleicacid; a capture probe oligomer as described herein for purifying theZika virus target nucleic acid; and a detection probe oligomer asdescribed herein for determining the presence or absence of a Zika virusamplification product. The reaction mixture may further include a numberof optional components such as, for example, arrays of capture probenucleic acids. For an amplification reaction mixture, the reactionmixture will typically include other reagents suitable for performing invitro amplification such as, e.g., buffers, salt solutions, appropriatenucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTPand UTP), and/or enzymes (e.g., reverse transcriptase, and/or RNApolymerase), and will typically include test sample components, in whicha Zika virus target nucleic acid may or may not be present. In addition,for a reaction mixture that includes a detection probe together with anamplification oligomer combination, selection of amplification oligomersand detection probe oligomers for a reaction mixture are linked by acommon target sequence (i.e., the reaction mixture will include a probethat binds to a sequence amplifiable by an amplification oligomercombination of the reaction mixture). Reaction mixtures may be aqueoussolutions or dried compositions (e.g., lyophilized compositions, spraydried compositions, etc.). Dried compositions may comprise buffers,bulking agents, and stabilizers. In one embodiment, a bulking agent ispresent in the dried composition, preferably at a concentration of lessthan about 5% (w/w). The bulking agent may be a disaccharide form of anamorphous sugar. Preferably, the bulking agent is one or more ofmannitol, trehalose sucrose, lactose, sorbitol, raffinose, and glucose.

Also provided by the subject invention are kits for practicing themethods as described herein. A kit in accordance with the presentinvention at least comprises one or more of the following: anamplification oligomer combination as described herein for amplificationof a Zika virus target nucleic acid; a capture probe oligomer asdescribed herein for purifying the Zika virus target nucleic acid; adetection probe oligomer as described herein for determining thepresence or absence of a Zika virus amplification product; and a probeprotection oligomer as described herein for detuning sensitivity of anassay for detecting the Zika virus target nucleic acid. The kits mayfurther include a number of optional components such as, for example,arrays of capture probe nucleic acids. Other reagents that may bepresent in the kits include reagents suitable for performing in vitroamplification such as, e.g., buffers, salt solutions, appropriatenucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTPand UTP), and/or enzymes (e.g., reverse transcriptase, and/or RNApolymerase). Oligomers as described herein may be packaged in a varietyof different embodiments, and those skilled in the art will appreciatethat the invention embraces many different kit configurations. Forexample, a kit may include amplification oligomers for only one targetsequence of a Zika virus genome, or it may include amplificationoligomers for multiple Zika virus target sequences. In addition, for akit that includes a detection probe together with an amplificationoligomer combination, selection of amplification oligomers and detectionprobe oligomers for a kit are linked by a common target sequence (i.e.,the kit will include a probe that binds to a sequence amplifiable by anamplification oligomer combination of the kit). In certain embodiments,the kit further includes a set of instructions for practicing methods inaccordance with the present invention, where the instructions may beassociated with a package insert and/or the packaging of the kit or thecomponents thereof.

Order of Steps

Target Capture: Nucleic acids underwent specimen processing and targetcapture prior to amplification essentially according to the proceduresdisclosed in published International Patent Application No.PCT/US2000/18685, except that templates were captured using Zika virustarget capture oligonucleotides having the target captureoligonucleotide sequences given herein. Notably, captureoligonucleotides do not participate in the amplification or detectionreactions of the assay. IVT or Virus-containing samples were combinedwith a target capture reagent to facilitate nucleic acid release andhybridization to capture oligonucleotides disposed on magnetic beads.Incubation was performed to capture Zika virus nucleic acids from thesample. Following the incubation, the magnetic beads and any capturetarget nucleic acids were transferred to a magnetic wash station for10-20 min. for a wash step. Captured target nucleic acids were thenassayed in an amplification reaction.

Transcription mediated amplification (TMA) reactions were carried outessentially as described in U.S. Pat. No. 5,399,491 (Kacian et al.).Isolated target nucleic acids were combined with primers inamplification reagent heated to 60° C. for 10 minutes and then cooled to42° C. to facilitate primer annealing. Enzyme reagent was then added tothe mixtures and the amplification reactions were carried out, as willbe familiar to those having an ordinary level of skill in the art.

Detection: After a one hour incubation at 42° C., the amplificationreaction volumes were subjected to hybridization assays employing probesinternally labeled with a chemiluminescent compound using techniquesfamiliar to those having an ordinary level of skill in the art, and thenused in amounts equivalent to about 2 E+06 to about 6 E+06 RLU for eachprobe in the hybridization reaction. (See e.g., U.S. Pat. Nos. 5,585,481and 5,639,604). Hybridization reactions were followed by addition of analiquot of 0.15 M sodium tetraborate (pH 8.5), and 1% TRITON X-100(Union Carbide Corporation; Danbury, Conn.). These mixtures were firstincubated at 60° C. for 10 minutes to inactivate the chemiluminescentlabel linked to unhybridized probe, and cooled briefly to roomtemperature (i.e., 15-30° C.) prior to reading the hybridization signal.Chemiluminescence due to hybridized probe in each sample was assayedusing commercially available instrumentation (Gen-Probe Incorporated;San Diego, Calif.) configured for injection of 1 mM nitric acid and 0.1%(v/v) hydrogen peroxide, followed by injection of a solution containing1 N sodium hydroxide. Results for the chemiluminescent reactions weremeasured in relative light units (RLU). In this procedure, thesignal/noise value corresponded to the chemiluminescent signal (measuredin RLU) generated by label associated with specifically hybridized probedivided by a background signal measured in the absence of a targetnucleic acid.

TABLE 2 Zika virus Assay Reagents Reagent Name Description InternalControl A HEPES buffered solution containing Reagent detergent and anRNA transcript. Target Capture A HEPES buffered solution containingReagent detergent, capture oligonucleotides and magnetic microparticles.Amplification Primers, dNTPs, NTPs and Reagent co-factors in TRISbuffered solution containing ProClin 300 as preservative. Enzyme MMLVReverse Transcriptase and Reagent T7 RNA Polymerase in HEPES/TRISbuffered solution containing 0.05% sodium azide as preservative. ProbeChemiluminescent oligonucleotide probes in Reagent succinate bufferedsolution containing detergent. IC Buffer A HEPES buffered solutioncontaining detergent.

The following are non-limiting examples of Zika virus amplification anddetection assays.

Example 1

Numerous sets of amplification and detection oligonucleotides wereconfigured to discriminately amplify and detect a zika virus. In thisexample, the transcription mediated amplification (TMA) reactions werecarried out essentially as described by Kacian et al., in U.S. Pat. No.5,399,491. Amplification reactions were conducted for variouscombinations of amplification oligonucleotides using about 5 pmoles perreaction of each T7 primer and nonT7 primer in a 75 uL amplificationreaction mixture. Amplification products were detected by hybridizationprotection assay (HPA) using about 2 10{circumflex over ( )}6 reactivelight units (RLU) per reaction of each of an acridinium ester(AE)-labeled detection probe.

A first set of amplification and detection oligonucleotides wereconfigured to amplify a target sequence within a zika virus region,wherein the zika virus region corresponds to nucleobases 7132 to 7641 ofSEQ ID NO:1 (zika07k region). The amplification oligomers were: for thenonT7 primers, SEQ ID NOs:11-23 & 27; and for the promoter primers, atarget-hybridizing sequence from SEQ ID NOs:113-115, & 125-134 joined attheir 3′ ends to the promoter primer sequence SEQ ID NO:179 (SEQ IDNOs:74-76, 78-83, 85-87, & 89). The detection probes wereesterase-labeled detection probes, SEQ ID NOs:69-73.

A second set of amplification and detection oligonucleotides wereconfigured to amplify a target sequence within a zika virus region,wherein the zika virus region corresponds to nucleobases 3340 to 3862 ofSEQ ID NO:1 (zika03k region). The amplification oligomers were: for thenonT7 primers, SEQ ID NOs:28-35; and for the promoter primers, atarget-hybridizing sequence from SEQ ID NOs:116, 117, & 135-138 joinedat their 3′ ends to the promoter primer sequence SEQ ID NO:179 (SEQ IDNOs:90-92, 94, 95, & 97). The detection probe was an esterase-labeleddetection probe, SEQ ID NO:58.

A third set of amplification and detection oligonucleotides wereconfigured to amplify a target sequence within a zika virus region,wherein the zika virus region corresponds to nucleobases 5806 to 6380 ofSEQ ID NO:1 (zika06k region). The amplification oligomers were: for thenonT7 primers, SEQ ID NOs:36-41, 43, 45, 46, & 48; and for the promoterprimers, a target-hybridizing sequence from SEQ ID NOs:98-106 joined attheir 3′ ends to the promoter primer sequence SEQ ID NO:179 (SEQ IDNOs:139-147). The detection probes were esterase-labeled detectionprobes, SEQ ID NOs:59 & 61-63.

In vitro transcripts (IVTs) were made for testing each set ofamplification and detection oligonucleotides (SEQ ID NOs:191-193,respectively). The stock concentration of each IVT was determined andthen the stock IVTs were each diluted to about 10 copies per mL.Separate target capture reactions were prepared, as described above, andusing the following target capture oligonucleotides: for capture of SEQID NO:191, SEQ ID NOs:160-163; for capture of SEQ ID NO:192, SEQ IDNOs:153-156; and for capture of SEQ ID NO:193, SEQ ID NOs:157-159.Captured IVT were then combined with an amplification reaction mixtureand an isothermal amplification reaction was performed. Amplificationoligomer pairs used for these tests were combinations of the non-T7primers and the promoter primers listed in each of the aboveamplification and detection oligomer sets. Amplification product wasdetected using an esterase-labeled detection probe. The detection probesSEQ ID NOs:69, 58, and 61 were used to detect amplification productsgenerated from the amplification oligomers of set one, set two, and setthree, respectively. Detection probe signal (RLU) was read using aluminometer. The RLU data for each reaction was then calculated againsta cutoff value to provide a signal to cutoff value (SCO). Tables 3-5,below, report the SCO data for combinations of amplification anddetection oligonucleotides from each of sets one to three.

TABLE 3 Set One Amplification and Detection Oligonucleotide CombinationsResults. SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 27 NO: 23 NO: 22 NO: 21 NO: 20NO: 19 NO: 18 NO: 17 NO: 16 NO: 15 NO: 14 NO: 13 NO: 12 NO: 11 SCO 88.82941.0 531.0 1687.1 1289.2 446.1 4304.3 374.3 725.5 337.1 978.8 469.8408.9 296.0 SEQ ID NO: 74 366.3 1000.6 349.4 3458.6 2993.7 174.2 0.4380.7 692.1 0.1 1929.4 195.6 4467.9 3328.0 SEQ ID NO: 75 439.5 824.3257.4 452.4 88.3 292.8 88.5 139.4 344.4 119.2 203.9 722.2 173.1 832.0SEQ ID NO: 76 1426.6 550.8 520.0 2965.9 396.6 233.9 617.0 239.5 1002.6206.1 0.3 302.2 536.5 SEQ ID NO: 78 997.5 910.2 1269.0 121.6 179.7 196.21082.9 1279.5 868.0 2519.8 2.7 3452.4 714.1 SEQ ID NO: 79 711.9 3103.7342.4 714.8 424.9 1262.0 714.2 1072.9 2047.3 740.9 578.7 604.2 378.2 SEQID NO: 80 8.0 11.9 12.0 1.0 4.6 2.4 1.2 5.0 1.1 0.4 0.1 0.3 3.3 SEQ IDNO: 81 3.9 46.7 17.6 6.9 8.3 1.2 33.8 1.0 3.7 6.7 2.2 11.2 4.7 SEQ IDNO: 82 77.3 2.2 619.6 166.1 1150.8 632.8 46.5 181.1 53.4 70.1 86.2 660.15.4 SEQ ID NO: 83 3.7 0.4 1.7 3.5 11.8 20.1 12.5 14.8 19.9 26.2 2.0 0.212.4 SEQ ID NO: 85 1.0 437.7 131.5 191.8 1694.7 370.5 15.3 387.1 387.7192.4 785.5 424.6 3032.3 SEQ ID NO: 86 363.1 433.1 1192.5 714.9 447.0296.6 1447.8 1034.7 1307.1 597.1 2176.1 1073.1 333.6 SEQ ID NO: 87

TABLE 4 Set Two Amplification and Detection Oligonucleotide CombinationsResults. SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SCO NO: 90 NO: 91 NO:92 NO: 94 NO: 95 NO: 97 SEQ ID NO: 28 1.9 5.0 369.4 5.6 241.7 28.3 SEQID NO: 29 0.6 0.5 412.6 197.6 684.8 15.9 SEQ ID NO: 30 1.3 21.4 182.9869.5 364.0 0.9 SEQ ID NO: 31 0.1 1.0 2930.5 934.7 1449.6 1239.5 SEQ IDNO: 32 2.1 1.9 724.4 7.9 675.3 174.9 SEQ ID NO: 33 1.0 1.4 179.9 0.2549.4 394.4 SEQ ID NO: 34 1.4 1.5 183.8 1.1 256.4 238.5 SEQ ID NO: 350.6 0.1 546.8 1.1 278.9 188.5

TABLE 5 Set Three Amplification and Detection OligonucleotideCombinations Results. SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: NO: NO:NO: SCO 102 103 104 105 106 SEQ ID NO: 36 0.5 1.6 2.6 2.4 1.2 SEQ ID NO:37 1.7 3.7 0.8 0.2 4.0 SEQ ID NO: 38 0.1 0.2 1.5 3.2 0.9 SEQ ID NO: 390.7 0.5 4.2 3.0 1.7 SEQ ID NO: 40 0.3 0.1 0.9 1.1 0.4 SEQ ID NO: 41 0.50.6 2.2 0.1 2.0 SEQ ID NO: 43 0.8 0.1 0.6 0.2 8.1 SEQ ID NO: 45 1.4 8.60.2 3.0 6.0 SEQ ID NO: 46 0.2 0.7 0.1 0.3 7.8 SEQ ID NO: 48 0.3 0.2 1.80.3 1.6

An SCO greater than 1 indicates that the amplification and detectionoligonucleotide combination provided a robust detection signal. A morepreferable SCO is greater than 10, and an even more preferable SCO isgreater than 100. These results indicate that various combinations ofoligonucleotides were effective in providing detectable zika virus IVTamplification product above a cutoff value. A number of theseamplification and detection oligonucleotide combinations were used infurther testing.

Example 2

This example describes multiplex amplification reactions using variousprimer sets for amplification of two Zika virus target sequences. TheZika virus assay involved three main steps, which take place in a singletube: sample preparation; Zika virus RNA target amplification byTranscription-Mediated Amplification (TMA); and detection of theamplification products (amplicon) by the Hybridization Protection Assay(HPA), as described above. Table 6 below lists the amplificationoligonucleotide combinations used in this assay.

TABLE 6 Zika virus Amplification Oligomers Condition Non T7 T7 1 SEQ IDNO: 16; SEQ ID NO: 78; SEQ ID NO: 11; & SEQ ID NO: 80; & SEQ ID NO: 30SEQ ID NO: 94 2 SEQ ID NO: 16; SEQ ID NO: 78; SEQ ID NO: 11; & SEQ IDNO: 80; & SEQ ID NO: 35 SEQ ID NO: 92 3 SEQ ID NO: 16; SEQ ID NO: 78;SEQ ID NO: 11; & SEQ ID NO: 75; & SEQ ID NO: 30 SEQ ID NO: 94 4 SEQ IDNO: 16; SEQ ID NO: 78; SEQ ID NO: 11; & SEQ ID NO: 75; & SEQ ID NO: 35SEQ ID NO: 92 5 SEQ ID NO: 12; SEQ ID NO: 79; SEQ ID NO: 17; & SEQ IDNO: 87; SEQ ID NO: 30 SEQ ID NO: 94; & SEQ ID NO: 95

Each of the conditions listed in Table 6 were tested in atranscription-mediated amplification (TMA) reaction using (1) Zika virusin vitro transcripts (IVT represented by SEQ ID NOs:191 & 192) each at10 and 0 copies per mL of internal control buffer (see example 3,below), and (2) a stock virus P6-740 (available from the Center forDisease Control, Atlanta, Ga.) at 0, 1 e-2 and 3 e-3 plaque formingunits per mL of BI0052 (Zika virus negative human serum). Transcriptionmediated amplification (TMA) reactions were carried out essentially asdescribed by Kacian et al., in U.S. Pat. No. 5,399,491. Amplificationreactions were conducted for various primer combinations using about 5pmoles per reaction of each T7 primer and nonT7 primer in a 75 uLamplification reaction mixture. Amplification products were detected byhybridization protection assay (HPA) using about 2 10{circumflex over( )}6 reactive light units (RLU) per reaction of each of an AE-labeleddetection probe having the nucleobase sequences shown in SEQ ID NOs:58 &69. Each condition was tested in replicates (5 replicates) for each ofthe IVT dilutions and each of the stock virus dilutions. Signal-to-noiseratios were calculated for each primer pair by dividing the RLU valueobserved at 10 copies of Zika virus IVT by the background RLU valueobserved at 0 copies of Zika virus IVT. Total RLU values for theconditions and dilutions are shown in Table 7 below.

TABLE 7 RLU Values of Zika virus T7/nonT7 Amplification OligoCombinations Condi- Condi- Condi- Condi- Condi- Target/ tion tion tiontion tion Dilution 1 2 3 4 5 Stock Virus 963 1,178 1,081 934 1,813 0 814955 1,328 2,386 1,473 pfu/mL 736 878 1,019 963 1,484 734 853 1,300 9181,402 1,415 759 861 826 1,429 Stock Virus 2,000,485 1,870,648 994,4601,355,632 1,929,586 1 1,941,247 1,927,834 1,713,798 1,818,677 1,957,834e-2 1,135,584 1,864,776 1,696,065 1,588,766 1,993,748 pfu/mL 1,930,0671,821,843 1,786,136 1,802,995 1,943,361 1,814,498 1,732,891 1,789,5711,729,585 1,838,143 Stock Virus 1,529,585 1,820,238 1,597,110 1,233,0042,038,343 3 1,048,528 1,920,906 1,225,351 1,851,168 2,026,636 e-31,027,851 1,875,564 1,091,207 1,080,080 2,043,965 pfu/mL 1,854,3261,887,128 1,870,477 1,711,219 2,056,585 998,130 1,905,529 981,5601,253,642 2,029,214 IVT 926 1,095 1,163 1,139 2,577 0 copies/mL 8842,375 1,075 994 1,350 806 1,014 1,112 972 1,527 850 1,398 1,061 9266,352 825 846 1,007 1,052 1,741 IVT #1 984,644 958,137 963,564 441,4592,079,278 (SEQ ID 1,034,198 924,170 952,185 586,993 410,577 NO: 191)968,755 1,812,164 927,418 1,606,937 643,748 10 945,882 907,020 896,725133,370 1,053,188 copies/mL 1,504 1,648,693 899,610 835,627 1,093,569IVT #2 1,076 746,827 28,652 359,399 2,075 (SEQ ID 978 943,480 666,687609,472 994,794 NO: 192) 1,022 30,263 1,128 947,261 972,426 10 1,133961,979 1,120 1,198 971,269 copies/mL 1,228 1,174 1,163 849,962 966,070

Primer pairs that demonstrated an RLU above 100,000 were considered tobe successful for amplification of Zika virus target nucleic acid to atleast as low as 10 copies of target nucleic acid per mL. These data showthat all of the amplification oligonucleotide combinations performedwell to amplify as low as 3 e-3 pfu per mL of stock zika virus and aslow as 10 copies per mL of IVT #1 (SEQ ID NO:191). Condition 1 failed toamplify and/or detect 10 copies of IVT #2 (SEQ ID NO:192), and condition3 successfully amplified and/or detected this target in only 1 out of 5replicates. Conditions 2, 4, & 5 combinations improved the number ofsuccessfully amplified and/or detected replicates, but still none showed5 of 5 successful amplification and detection reactions. Thus, thecombinations of just amplification oligomers SEQ ID NOs:30 and 94performed poorly in this set of tests (conditions 1 and 3), however,adding SEQ ID NO:95 to the combination resulted in a very goodperforming assay (condition 5). Similarly, the combination ofamplification oligonucleotides SEQ ID NOs:35 & 92 performed well inthese tests 3 and 4 out of 5 (conditions 2 and 4). Further combinationswere tested.

Example 3

This example describes Zika virus amplification and detection assaysperformed using different oligomer combinations. Sample andoligonucleotide combinations are listed in Tables 8-9 below.Amplification and detection reactions were performed using the HologicPanther System (Hologic, Inc., Marlborough, Mass.).

TABLE 8 Zika virus-specific Oligonucleotides Condition Non T7 T7 1 SEQID NO: 12; SEQ ID NO: 79; SEQ ID NO: 17; & SEQ ID NO: 87; SEQ ID NO: 30SEQ ID NO: 94; & SEQ ID NO: 95 2 SEQ ID NO: 12; SEQ ID NO: 87; SEQ IDNO: 17; & SEQ ID NO: 89 SEQ ID NO: 30 SEQ ID NO: 95 3 SEQ ID NO: 12; SEQID NO: 79; SEQ ID NO: 17; & SEQ ID NO: 89 SEQ ID NO: 94; & SEQ ID NO: 30SEQ ID NO: 95 4 SEQ ID NO: 16; SEQ ID NO: 78; SEQ ID NO: 11; & SEQ IDNO: 80; & SEQ ID NO: 35 SEQ ID NO: 92 5 SEQ ID NO: 16; SEQ ID NO: 78;SEQ ID NO: 11; & SEQ ID NO: 89; & SEQ ID NO: 35 SEQ ID NO: 92 6 SEQ IDNO: 17; SEQ ID NO: 18; SEQ ID NO: 74; & SEQ ID NO: 30; & SEQ ID NO: 95SEQ ID NO: 35

TABLE 9 Samples Tested Sample Identifier Sample Description A IVT#1 Zikavirus In Vitro Transcript (IVT) (SEQ ID NO: 191) in IC buffer (Table 2)B IVT#2 Zika virus In Vitro Transcript (IVT) (SEQ ID NO: 192) in ICbuffer (Table 2) C Virus P6-740 Stock virus in BI0052 serum D Donatedblood Zika virus spiked blood samples diluted in human plasma E ClinicalSamples Plasma samples from Zika virus positive donors

A multiplex TMA amplification and detection reaction was performed todetermine the analytical sensitivity of a combination of amplificationand detection oligomers. This Zika virus assay involved three mainsteps, which take place in a single tube: sample preparation; Zika virusRNA target amplification by Transcription-Mediated Amplification (TMA);and detection of the amplification products (amplicon) by theHybridization Protection Assay (HPA), as described above. Tested samplesincluded in vitro transcripts (IVTs) serially diluted in IC buffer,donated human blood spiked with Zika virus RNA and serially diluted inhuman plasma, cadaveric plasma or cadaveric serum spiked with Zika virusRNA, and clinical samples determined to be positive for Zika virus.Transcription mediated amplification (TMA) reactions were carried outessentially as described by Kacian et al., in U.S. Pat. No. 5,399,491.Amplification reactions were conducted for various combinations ofamplification oligonucleotides using about 5 pmoles per reaction of eachT7 primer and nonT7 primer in a 75 uL amplification reaction mixture.Amplification products were detected by hybridization protection assay(HPA) using about 2 10{circumflex over ( )}6 reactive light units (RLU)per reaction of each of an acridinium ester (AE)-labeled detectionprobe. Each amplification oligonucleotide condition was tested inreplicates of 5 on each of the sample dilutions. Amplification oligomercombinations are shown in Table 8, above. Detection reactions wereperformed using SEQ ID NOs:58 & 69 as AE-labeled detection probeoligomers. Tables 10-12 show the results of these reactions. Table 10shows the RLU values for conditions 1-5 of amplificationoligonucleotides. Table 11 and Table 12 show the analytical sensitivityand clinical sensitivity for condition 6 of amplificationoligonucleotides. In Table 11 average means average of the reactivesamples unless all samples were non-reactive in which case average meansaverage of all non-reactive samples.

TABLE 10 RLU Values of Zika virus T7/nonT7 Amplification OligoCombinations Condi- Condi- Condi- Condi- Condi- Sample Type tion tiontion tion tion Copies/mL 1 2 3 4 5 A 48,757 757,559 762,192 9,523781,594 10 375,358 8,702 418,044 63,009 267,402 copies/mL 1,078 776,747798,691 354,815 807,916 758,423 800,552 758,118 27,867 791,362 746,033304,704 763,195 308,653 761,598 B 906 1,930 705,223 126,982 455,773 1025,945 716,734 1,219 4,289 2,859 copies/mL 1,002 4,320 713,626 82,8633,843 373,889 6,343 15,160 2,831 2,106 1,246 3,818 1,186 2,303 775 C4,491 1,287 3,077 6,013 1,459 1 e-2 3,012 1,102 2,844 6,269 11,925pfu/mL 6,754 957 2,294 1,772 4,123 7,372 1,538 1,230 682 1,237 5,1396,444 676,451 1,352 783 C 4,058 10,823 17,014 4,723 1,134 3 e-3 98716,253 2,328 1,416 2,029 pfu/mL 1,527 10,552 3,119 3,592 2,732 4,58624,009 922,487 3,562 795 1,170 6,060 3,090 744 9,447 C 944 1,074 1,0464,074 5,482 0 pfu/mL 11,680 880 3,649 4,061 2,660 6,761 2,365 3,05918,396 3,426 7,895 1,186 9,854 2,312 1,513 3,503 5,757 12,162 16,50411,853

TABLE 11 Analytical Sensitivity of a Multiplexed Amplification andDetection Reaction Sample Type(s) #Reactive/ % Reactive SCO Copies/mLTested (95% CI) Average % cv A&B 20/20 100 (84-100) 33.5 4.3% 90Copies/mL A&B 72/72 100 (95-100) 33.2 4.7% 30 Copies/mL A&B 66/72 92(83-96) 32.0 13.6% 10 Copies/mL A&B 39/72 54 (43-65) 28.7 23.3% 3Copies/mL A&B 16/72 22 (14-33) 27.7 26.4% 1 Copy/mL A&B  2/72 3 (1-10)24.5 50.7% 0.3 Copies/mL D 20/20 100 (84-100) 32.5 3.3% 90 Copies/mL D72/72 100 (95-100) 32.6 4.1% 30 Copies/mL D 72/72 100 (95-100) 31.5 9.7%10 Copies/mL D 62/72 86 (76-92) 25.8 29.3% 3 Copies/mL D 27/72 38(27-49) 24.1 37.1% 1 Copy/mL D 14/72 19 (12-30) 17.7 32.8% 0.3 Copies/mLD  1/72 1 (0-8) 15.7 N/A 0.1 Copies/mL No Template  0/144 0 (0-5) 0.0N/A 0 Copies/mL

TABLE 12 Clinical Sensitivity of a Multiplexed Amplification andDetection Reaction Sample Neat Pooled Origin # Reactive / % # Reactive /% Country Tested Reactive SCO Tested Reactive SCO Colombia 1/1 100% 30.54/4 100% 30.7 Colombia 1/1 100% 31.3 4/4 100% 31.8 Colombia 1/1 100%31.3 4/4 100% 31.6 Colombia 1/1 100% 32.5 4/4 100% 19.7 Colombia 1/1100% 32.8 4/4 100% 28.1 Colombia 1/1 100% 32.5 4/4 100% 31.4 Colombia1/1 100% 31.2 4/4 100% 30.8 Colombia 1/1 100% 29.8 4/4 100% 31.1Colombia 1/1 100% 32.1 3/4  75% 25.9 Dominican 1/1 100% 30.9 4/4 100%31.5 Republic Dominican 1/1 100% 31.8 4/4 100% 27.0 Republic Dominican1/1 100% 32.3 4/4 100% 31.7 Republic Dominican 1/1 100% 32.1 4/4 100%30.5 Republic Dominican 1/1 100% 30.6 4/4 100% 31.2 Republic Dominican1/1 100% 31.8 1/4 25% 16.2 Republic Dominican 1/1 100% 33.4 4/4 100%27.5 Republic Dominican 1/1 100% 30.3 4/4 100% 31.3 Republic Dominican1/1 100% 31.0 4/4 100% 22.8 Republic Dominican 1/1 100% 32.1 4/4 100%30.9 Republic Dominican 1/1 100% 31.7 4/4 100% 31.9 Republic Dominican1/1 100% 32.0 4/4 100% 32.5 Republic Dominican 1/1 100% 29.6 4/4 100%31.6 Republic Dominican 1/1 100% 29.8 4/4 100% 31.6 Republic Colombia1/1 100% 30.3 4/4 100% 31.7 Colombia 1/1 100% 31.7 4/4 100% 32.2Colombia 1/1 100% 29.6 4/4 100% 30.2

These data show that conditions 3 and 5 performed well at amplifying aslow as 10 copies per mL of IVT #1 (SEQ ID NO:191). Conditions 1, 2 and 4amplified 2 to 3 of 5 replicates containing 10 copies per mL of SEQ IDNO;191. Conditions 1, 2, 3 & 5 amplifies 1 to 2 out of 5 replicatescontaining 10 copies per mL of IVT #2 (SEQ ID NO:192). In thisexperiment, none of the conditions performed well at amplifying targetnucleic acid from various pfu/mL of P6-740 virus, with only conditions 3amplifying 1 of 5 replicates at each concentration. Probit analysis ofthe multiplexed assay showed detection probabilities in copies/mL (95%fiducial limits) for the IVTA&B and spiked blood donor samples to be (i)204 (2.0-2.9) for 50% probability and 13.4 (9.9-20.3) for 95%probability, and (ii) 1.0 (0.8-1.2) for 50% probability, and 5.9(4.3-8.9) for 95% probability. Clinical samples showed 100% (95% CI87-100%) sensitivity (26 of 26) in neat samples and 96% (95% CI 91-98%)sensitivity (100 of 104) in 1:16 pooled samples. Thus, theseamplification oligonucleotide combinations demonstrate very sensitivedetection of Zika virus RNA down to about 6 to about 13 copies of Zikavirus nucleic acid per mL for an assay specificity of greater than99.90%.

Sequences

TABLE 13 Exemplary Oligomer Sequences, Reference Sequences, and RegionsSEQ ID NO: SEQUENCE (5′ TO 3′)   1 AY632535.2 GI:226374362¹   2TCYCTTGGAGTGCTTGTGA   3 TCYCTTGGAGTGCTTGTGATT   4TCYCTTGGAGTGCTTGTGATTYT   5 TCYCTTGGAGTGCTTGTGATTY   6YCCYAAYAAACCTGGAGATGAGTA   7 AAYAAACCTGGAGATG   8 YAAYAAACCTGGAGATG   9TGGCTTGAAGCAAGAATGCT  10 GCTTGAAGCAAGAAT  11 AGGACAGCAGCTGGCATCAT  12AGGACGGCAGCTGGCATCAT  13 AGGACAGCAGCTGGCATCATG  14 AGGACGGCAGCTGGCATCATG 15 AGGACAGCAGCTGGCATCATGA  16 AGGACGGCAGCTGGCATCATGA  17AGGACAGCAGCTGGCATCATGAA  18 AGGACGGCAGCTGGCATCATGAA  19ACGGCAGCTGGCATCATGAA  20 GACGGCAGCTGGCATCATGAA  21ACAGCAGCTGGCATCATGAAGAA  22 AGGACAGCAGCTGGCATCATGAAGAA  23AGAACAGCAGCTGGCATCATGAAGAA  24 AGRACRGCAGCTGGCATCATGAAGAA  25ACRGCAGCTGGCATCAT  26 RACRGCAGCTGGCATCATGAA  27 GTTGTGGATGGAATAGTGGT  28TCTCTTGGAGTGCTTGTGATTC  29 TCTCTTGGAGTGCTTGTGATT  30 TCTCTTGGAGTGCTTGTGA 31 TCTCTTGGAGTGCTTGTGATTCT  32 TCCCTTGGAGTGCTTGTGATTCT  33TCCCTTGGAGTGCTTGTGATTC  34 TCCCTTGGAGTGCTTGTGATT  35 TCCCTTGGAGTGCTTGTGA 36 AACAAACCTGGAGATGAGTA  37 CAACAAACCTGGAGATGAGTA  38AACAAACCTGGAGATGAGT  39 CAACAAACCTGGAGATGAGT  40 CAATAAACCTGGAGATGAGT 41 CCCAATAAACCTGGAGATGAGT  42 CCYAAYAAACCTGGAGATGAGTA  43CCCAACAAACCTGGAGATGAGTA  44 YCCYAAYAAACCTGGAGATG  45TCCTAACAAACCTGGAGATG  46 CCCAACAAACCTGGAGATGAGT  47CCYAAYAAACCTGGAGATGAG  48 CCCAACAAACCTGGAGATGAG  49AGRACRGCAGCTGGCATCATGAAGA  50 AGRACRGCAGCTGGCATCATGAAGAA  51AGRACRGCAGCTGGCATCATGA  52 AGRACRGCAGCTGGCATCAT  53AGRACRGCAGCTGGCATCATGAA  54 TRAAGAAGAGAATGACCAC  55 URAAGAAGAGAAUGACCAC 56 GTTGTGGAKGGAATAGTGGT  57 GUUGUGGAKGGAAUAGUGGT  58UGAAGAAGAGAAUGACCAC  59 UGUAUGGAGGUGGGUGUGC  60 CUGGCUUGAAGCAAGAAUGCT 61 UGGCUUGAAGCAAGAAUGCT  62 UGGCUUGAAGCAAGAAT  63 GCUUGAAGCAAGAAUGCT 64 CATTGACACAATG  65 ACTGACATTGACACAATGACWAT  66ACUGACAUUGACACAAUGACWAT  67 GACAUUGACACAAUGACWAT  68 CAUUGACACAAUGACWAT 69 GUUGUGGAUGGAAUAGUGGT  70 ACUGACAUUGACACAAUGACAAT  71ACUGACAUUGACACAAUG  72 GACAUUGACACAAUGACAAT  73 CAUUGACACAAUGACAAT  74AATTTAATACGACTCACTATAGGGAGAGTCATTGTGTCAATGTCAG  75AATTTAATACGACTCACTATAGGGAGAGTCATTGTGTCAATGTCAGT  76AATTTAATACGACTCACTATAGGGAGATCATTGTGTCAATGTCAGT  77AATTTAATACGACTCACTATAGGGAGAGTCWATWGTCATTGTGTCA  78AATTTAATACGACTCACTATAGGGAGAGTCTATTGTCATTGTGTCA  79AATTTAATACGACTCACTATAGGGAGAGTCTATTGTCATTGTGT  80AATTTAATACGACTCACTATAGGGAGAATTGTCATTGTGTCAATGTCAGT  81AATTTAATACGACTCACTATAGGGAGAATTGTCATTGTGTCAATGTCA  82AATTTAATACGACTCACTATAGGGAGAATTGTCATTGTGTCAATGTC  83AATTTAATACGACTCACTATAGGGAGAATTGTCATTGTGTCAATGT  84AATTTAATACGACTCACTATAGGGAGAYTGYCCCATCTTYTTYT  85AATTTAATACGACTCACTATAGGGAGATTGTCCCATCTTCTTCT  86AATTTAATACGACTCACTATAGGGAGAGTAACACTTGTCCCATCTT  87AATTTAATACGACTCACTATAGGGAGACTGCTATGAGTAACACTTGTCCCATCTT  88AATTTAATACGACTCACTATAGGGAGAGTCWATWGTCATTGTGTCAATGTCAG  89AATTTAATACGACTCACTATAGGGAGAGTCTATTGTCATTGTGTCAATGTCAG  90AATTTAATACGACTCACTATAGGGAGACTACCAGCACTGCCATTGATGTGCT  91AATTTAATACGACTCACTATAGGGAGACCAGCACTGCCATTGATGTGCT  92AATTTAATACGACTCACTATAGGGAGACTACCAGCACTGCCATTGATGT  93AATTTAATACGACTCACTATAGGGAGATRRCTACCAGCACTGCCAT  94AATTTAATACGACTCACTATAGGGAGATAGCTACCAGCACTGCCAT  95AATTTAATACGACTCACTATAGGGAGACTACCAGCACTGCCATTGATGTGC  96AATTTAATACGACTCACTATAGGGAGATRRCTACCAGCACTGCCATTG  97AATTTAATACGACTCACTATAGGGAGATAGCTACCAGCACTGCCATTG  98AATTTAATACGACTCACTATAGGGAGAAGCATTCTTGCTTCAAGCCA  99AATTTAATACGACTCACTATAGGGAGAGCATTCTTGCTTCAAGCCA 100AATTTAATACGACTCACTATAGGGAGAGGAGCATTCTTGCTTCAAGCCA 101AATTTAATACGACTCACTATAGGGAGAGTCAAGAAGCATTCTTGCTTCA 102AATTTAATACGACTCACTATAGGGAGATAGAGCGAGGCTATGAGGCCATC 103AATTTAATACGACTCACTATAGGGAGATAGAGCGAGGCTATGAGGCCAT 104AATTTAATACGACTCACTATAGGGAGATAGAGCGAGGCTATGAGGCCA 105AATTTAATACGACTCACTATAGGGAGATAGAGCGAGGCTATGAGG 106AATTTAATACGACTCACTATAGGGAGATAGAGCGAGGCTATGAG 107AATTTAATACGACTCACTATAGGGAGAGTCWATWGTCATTGTGT 108AATTTAATACGACTCACTATAGGGAGACTGCYATGAGTARCACYTGYCCCATCTT 109AATTTAATACGACTCACTATAGGGAGAGRAGCATTCTTGCTTCAAGCCA 110AATTTAATACGACTCACTATAGGGAGAGTCAAGRAGCATTCTTGCTTCA 111TRRCTACCAGCACTGCCAT 112 GTCWATWGTCATTGTGT 113 GTCATTGTGTCAATGTCAG 114GTCTATTGTCATTGTGT 115 CTGCTATGAGTAACACTTGTCCCATCTT 116TAGCTACCAGCACTGCCAT 117 CTACCAGCACTGCCATTGATGTGC 118CTGCYATGAGTARCACYTGYCCCATCTT 119 GTCWATWGTCATTGTGTCA 120YTGYCCCATCTTYTTYT 121 GTCWATWGTCATTGTGTCAATGTCAG 122TRRCTACCAGCACTGCCATTG 123 GRAGCATTCTTGCTTCAAGCCA 124GTCAAGRAGCATTCTTGCTTCA 125 GTCATTGTGTCAATGTCAGT 126 TCATTGTGTCAATGTCAGT127 GTCTATTGTCATTGTGTCA 128 ATTGTCATTGTGTCAATGTCAGT 129ATTGTCATTGTGTCAATGTCA 130 ATTGTCATTGTGTCAATGTC 131 ATTGTCATTGTGTCAATGT132 TTGTCCCATCTTCTTCT 133 GTAACACTTGTCCCATCTT 134GTCTATTGTCATTGTGTCAATGTCAG 135 CTACCAGCACTGCCATTGATGTGCT 136CCAGCACTGCCATTGATGTGCT 137 CTACCAGCACTGCCATTGATGT 138TAGCTACCAGCACTGCCATTG 139 AGCATTCTTGCTTCAAGCCA 140 GCATTCTTGCTTCAAGCCA141 GGAGCATTCTTGCTTCAAGCCA 142 GTCAAGAAGCATTCTTGCTTCA 143TAGAGCGAGGCTATGAGGCCATC 144 TAGAGCGAGGCTATGAGGCCAT 145TAGAGCGAGGCTATGAGGCCA 146 TAGAGCGAGGCTATGAGG 147 TAGAGCGAGGCTATGAG 148CCAGCACTGCCAT 149 CTACCAGCACTGCCAT 150 GTCWATWGTCATTGTGTCAATGTCAGT 151CTGCYATGAGTARCACYTGYCCCATCTTYTTYTCCACYTGGGGGTCWATWGTCATTGT GTCAATGTCAGT152 TRRCTACCAGCACTGCCATTGATGTGC 153GCCUUAUCUCCAUUCCAUACCATTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 154GGCCUUAUCUCCAUUCCAUACCATTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 155ACAUCUCCUCCAGUGUUCAUUUCTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 156AGCCACAUCUCCUCCAGUGUUCATTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 157AUGACUCUCUCACCAUCAAGUAUGACTTAAAAAAAAAAAAAAAAAAAAAAAAAAAA AA 158AGCUUGAACUCUCCCUCAAUGGCGGTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAA AA 159CUGUCUUCCAUUAUGGUGUUGUUGGTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAA AA 160CCUGGGAUCAAGUACAUGUAGUGTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 161CCUGGGAUCAAGUACAUGUAGTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 162GAGGAGUUCCAGUAUUUGUUUGGTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 163GAGGAGUUCCAGUAUUUGUUUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 164CGGCCAAUCAGUUCAUCUUGGUGGCGGCTTTAAAAAAAAAAAAAAAAAAAAAAAAA AAAAA 165CAGCUAGUCUCCAGUUCAGGCCCTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 166CAGCUAGUCUCCAGUUCAGGCCC 167 CGGCCAAUCAGUUCAUCUUGGUGGCGGC 168GGCCUUAUCUCCAUUCCAUACCA 169 ACAUCUCCUCCAGUGUUCAUUUC 170CCUGGGAUCAAGUACAUGUAG 171 GCCUUAUCUCCAUUCCAUACCA 172AGCCACAUCUCCUCCAGUGUUCA 173 AUGACUCUCUCACCAUCAAGUAUGACTT 174AGCUUGAACUCUCCCUCAAUGGCGG 175 CUGUCUUCCAUUAUGGUGUUGUUGG 176CCUGGGAUCAAGUACAUGUAGUG 177 GAGGAGUUCCAGUAUUUGUUUGG 178GAGGAGUUCCAGUAUUUGUUU 179 AATTTAATACGACTCACTATAGGGAGA 180 TTTTTTTTTTTTTT181 TTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 182 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA183 AGGACAGCAGCTGGCATCATGAAGAATCCCGTTGTGGATGGAATAGTGGTAACTGACATTGACACAATGACAATAGACCCCCAGGTGGAGAAGAAGATGGGACAAGTGTTACTCAT AGCAG 184AGGACAGCAGCTGGCATCATGAAGAATCCCGTTGTGGATGGAATAGTGGTAACTGACATTGACACAATGACAATAGAC 185CCCTAACAAACCTGGAGATGAGTACATGTATGGAGGTGGGTGTGCAGAGACTGATGAAGGCCATGCACACTGGCTTGAAGCAAGAATGCTTCTTGACAACATCTACCTCCAGGATGGCCTCATAGCCTCGCTCTA 186TCTCTTGGAGTGCTTGTGATTCTACTCATGGTGCAGGAGGGGTTGAAGAAGAGAATGACCACAAAGATCATCATGAGCACATCAATGGCAGTGCTGGTAGTCA 187TCYCTTGGAGTGCTTGTGATTYTRCTCATGGTGCAGGARGGGYTRAAGAAGAGAATGACCACAAAGATCATCATRAGCACATCAATGGCAGTGCTGGTAGYYA 188YCCYAAYAAACCTGGAGATGAGTAYMTGTATGGAGGTGGGTGYGCAGAGACTGAYGAAGRYCAYGCACACTGGCTTGAAGCAAGAATGCTYCTTGACAAYATYTACCTCCARGATGGCCTCATAGCYTCGCTCTA 189AGRACRGCAGCTGGCATCATGAAGAAYCCYGTTGTGGAKGGAATAGTGGTRACTGACATTGACACAATGACWATWGAC 190AGRACRGCAGCTGGCATCATGAAGAAYCCYGTTGTGGAKGGAATAGTGGTRACTGACATTGACACAATGACWATWGACCCCCARGTGGARAARAAGATGGGRCARGTGYTACTCA TRGCAG 191GGGCGAAUUGGGUACCGGGCCCCCCCUCGAGGUCGACGGUAUCGAUAAGCUUGCUGCUAAUGAUGGGUUGCUAUUCACAAUUAACACCCCUGACUCUGAUAGUAGCUAUCAUUCUGCUUGUGGCGCACUACAUGUACUUGAUCCCAGGCCUACAAGCGGCAGCAGCGCGUGCUGCCCAGAAAAGGACAGCAGCUGGCAUCAUGAAGAAUCCCGUUGUGGAUGGAAUAGUGGUAACUGACAUUGACACAAUGACAAUAGACCCCCAGGUGGAGAAGAAGAUGGGACAAGUGUUACUCAUAGCAGUAGCCAUCUCCAGUGCUGUGCUGCUGCGGACCGCCUGGGGAUGGGGGGAGGCUGGAGCUCUGAUCACAGCAGCGACCUCCACCUUGUGGGAAGGCUCUCCAAACAAAUACUGGAACUCCUCUACAGCCACCUCACUGUGCAACAUCUUCAGAGGAAGCUAUCUGGCAGGAGCUUCCCUUAUCUAUACAGUGACGAGAAACGCUGA AUU 192GGGCGAAUUGGGUACCGGGCCCCCCCUCGAGGUCGACGGUAUCGAUAAGCUUGAUAUCGAAUUCCUGCAGCCCGGGGGAUCCCACUAUCGUUUCGAGCAAAAGACGGCUGCUGGUAUGGAAUGGAGAUAAGGCCCAGGAAAGAACCAGAGAGCAACUUAGUGAGGUCAAUGGUGACAGCGGGGUCAACCGAUCAUAUGGACCACUUCUCUCUUGGAGUGCUUGUGAUUCUACUCAUGGUGCAGGAGGGGUUGAAGAAGAGAAUGACCACAAAGAUCAUCAUGAGCACAUCAAUGGCAGUGCUGGUAGUCAUGAUCUUGGGAGGAUUUUCAAUGAGUGACCUGGCCAAGCUUGUGAUCCUGAUGGGUGCUACUUUCGCAGAAAUGAACACUGGAGGAGAUGUAGCUCACUUGGCAUUGGUAGCGGCAUUUAAAGUCAGACCAGCCUUGCUGGUCUCCUUCAUUUUCAGAGCCAAUUGGACACCCCGUGAGAGCAUGCUGCUAGCGGC C 193GGGCGAAUUGGGUACCGGGCCCCCCCUCGAGGUCGACGGUAUCGAUAAGCUUGUCAUAGACUCUAGGAGAUGCCUAAAACCAGUCAUACUUGAUGGUGAGAGAGUCAUCUUGGCUGGGCCCAUGCCUGUCACGCAUGCUAGUGCUGCUCAGAGGAGAGGACGUAUAGGCAGGAACCCUAACAAACCUGGAGAUGAGUACAUGUAUGGAGGUGGGUGUGCAGAGACUGAUGAAGGCCAUGCACACUGGCUUGAAGCAAGAAUGCUUCUUGACAACAUCUACCUCCAGGAUGGCCUCAUAGCCUCGCUCUAUCGGCCUGAGGCCGAUAAGGUAGCCGCCAUUGAGGGAGAGUUUAAGCUGAGGACAGAGCAAAGGAAGACCUUCGUGGAACUCAUGAAGAGAGGAGACCUUCCCGUCUGGCUAGCCUAUCAGGUUGCAUCUGCCGGAAUAACUUACACAGACAGAAGAUGGUGCUUUGAUGGCACAACCAACAACACCAUAAUGGAAGACAGCGUACCAGCAGAGGUGUGGACAAAGUAUGGAGAGAAGAGAGUGCUCAAACCG AAUU 194AGRACRGCAGCTGGCATCATGAAGAAYCCYGTTGTGGAKGGAATAGTGGTRACTGACA TTGACACAATGAC195 YMTGTATGGAGGTGGGTGYGCAGAGACTGAYGAAGRYCAYGCACACTGGCTTGAAGCAAGAATGCTYCTTGACAAYATYTACCTCCAR 196RCTCATGGTGCAGGARGGGYTRAAGAAGAGAATGACCACAAAGATCATCATR 197YCCYGTTGTGGAKGGAATAGTGGTR 198YCCYGTTGTGGAKGGAATAGTGGTRACTGACATTGACACAATGACWATWGACCCCCA RGTGGA ¹SEQ IDNO: 1 is referenced to its GenBank Accession Number and Version.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims. All publications, patents, andpatent applications cited herein are hereby incorporated by reference intheir entireties for all purposes.

What is claimed is:
 1. A combination of at least two amplificationoligomers for amplifying a Zika virus nucleic acid in a sample, theoligomer combination comprising: at least two amplification oligomersconfigured to amplify a target sequence corresponding to a Zika virustarget nucleic acid, wherein the target sequence consists of SEQ IDNO:187, the RNA equivalent of SEQ ID NO:187, or the DNA or RNAcomplement of SEQ ID NO:187, wherein the first amplification oligomerand the second amplification oligomer are configured to hybridize toopposite ends of the target sequence to generate amplification products;wherein the first amplification oligomer comprises a target-hybridizingsequence that is from 19 to 23 contiguous nucleobases in length, andwherein the second amplification oligomer comprises a target-hybridizingsequence that is from 19 to 25 contiguous nucleobases in length, whereinthe second amplification oligomer target-hybridizing sequence is joinedat its 5′ end to a T7 promoter sequence.
 2. The combination of claim 1,wherein the first amplification oligomer target-hybridizing sequenceconsists of from 19 to 23 contiguous nucleobases of SEQ ID NO:4.
 3. Thecombination of claim 2, wherein said contiguous nucleobases of SEQ IDNO:4 include SEQ ID NO:2.
 4. The combination of claim 3, wherein thefirst amplification oligomer target-hybridizing sequence is selectedfrom the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and SEQ IDNO:35.
 5. The combination of claim 4, wherein the second amplificationoligomer target-hybridizing sequence is selected from the groupconsisting of SEQ ID NO:117 and SEQ ID NO:137.
 6. The combination ofclaim 1, wherein the second amplification oligomer target-hybridizingsequence consists of from 19 to 25 contiguous nucleobases of SEQ IDNO:152.
 7. The combination of claim 6, wherein said contiguousnucleobases of SEQ ID NO:152 include SEQ ID NO:148 or SEQ ID NO:149. 8.The combination of claim 7, wherein the second amplification oligomertarget-hybridizing sequence is selected from the group consisting of SEQID NO:111, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:122, SEQ ID NO:135,SEQ ID NO:136, SEQ ID NO:137, and SEQ ID NO:138.
 9. The combination ofclaim 8, wherein the nucleotide sequence of the second amplificationoligomer is selected from the group consisting of SEQ ID NO:90, SEQ IDNO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ IDNO:96, and SEQ ID NO:97.
 10. The combination of claim 1, wherein the T7promoter sequence consists of SEQ ID NO:179.
 11. An amplificationreaction mixture, comprising the at least two amplification oligomers ofclaim 1, wherein (i) the mixture is a buffered aqueous solution; or (ii)the mixture is a dried composition.
 12. A kit comprising the at leasttwo amplification oligomers of claim
 1. 13. A method for determining thepresence or absence of a Zika virus nucleic acid in a sample, the methodcomprising the steps of: (A) contacting a sample with a combination ofat least two amplification oligomers configured to amplify a targetsequence corresponding to a Zika virus target nucleic acid, wherein thetarget sequence consists of SEQ ID NO:187, the RNA equivalent of SEQ IDNO:187, or the DNA or RNA complement of SEQ ID NO:187, wherein the firstamplification oligomer and the second amplification oligomer areconfigured to hybridize to opposite ends of the target sequence togenerate amplification products; wherein the first amplificationoligomer comprises a target-hybridizing sequence that is from 19 to 23contiguous nucleobases in length, and wherein the second amplificationoligomer comprises a target-hybridizing sequence that is from 19 to 25contiguous nucleobases in length, wherein the second amplificationoligomer target-hybridizing sequence is joined at its 5′ end to a T7promoter sequence; (B) performing an in vitro nucleic acid amplificationreaction, wherein any Zika virus target nucleic acid present in thesample is used as a template for generating an amplification product;and (C) detecting the presence or absence of the amplification product,thereby determining the presence or absence of the Zika virus nucleicacid in the sample.
 14. The method of claim 13, wherein the firstamplification oligomer target-hybridizing sequence consists of from 19to 23 contiguous nucleobases of SEQ ID NO:4.
 15. The method of claim 14,wherein said contiguous nucleobases of SEQ ID NO:4 include SEQ ID NO:2.16. The method of claim 15, wherein the first amplification oligomertarget-hybridizing sequence is selected from the group consisting of SEQID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, and SEQ ID NO:35.
 17. The method of claim 13,wherein the second amplification oligomer target-hybridizing sequenceconsists of from 19 to 25 contiguous nucleobases of SEQ ID NO:152. 18.The method of claim 17, wherein said contiguous nucleobases of SEQ IDNO:152 include SEQ ID NO:148 or SEQ ID NO:149.
 19. The method of claim18, wherein the second amplification oligomer target-hybridizingsequence is selected from the group consisting of SEQ ID NO:111, SEQ IDNO:116, SEQ ID NO:117, SEQ ID NO:122, SEQ ID NO:135, SEQ ID NO:136, SEQID NO:137, and SEQ ID NO:138.
 20. The method of claim 19, wherein thenucleotide sequence of the second amplification oligomer is selectedfrom the group consisting of SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92,SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, and SEQ IDNO:97.